Particular agents

ABSTRACT

A novel means of pharmaceutical delivery for therapy or prophylaxis or to assist surgical or diagnostic operations on the living body is provided by agents which undergo neuronal endocytosis and axonal transport following pharmaceutical administration into vascularized, peripherally innervated tissue, e.g., intramuscular injections of a nerve adhesion molecule comprising a physiologically active substance or a diagnostic marker.

This application is a continuation of U.S. patent application Ser. No.08/473,697, filed on Jun. 7, 1995, which issued as U.S. Pat. No.5,948,384 on Sep. 7, 1999, which is a divisional of U.S. patentapplication Ser. No. 07/988,919, filed on Apr. 5, 1993 now abandoned,which is a 371 of International Application No. PCT/EP91/01780, filed onSep. 13, 1991.

This invention relates to novel particulate agents for use indiagnostics and therapy, especially in diagnostic imaging, and moreparticularly diagnostic imaging or therapeutic treatment via the neuralsystem.

In the living body, pain, paralysis and neural dysfunction can beinferred from electrical studies such as EMG, NCV and SSEP, but thesekinds of assessment have continued to prove awkward and imprecise. WhileCT and MRI have made it possible to diagnose a wide variety ofstructural problems affecting the brain and spinal chord, and whilestudies on excised tissue and post mortem studies have enabled neuronalpathways to be traced, there is currently virtually no effective meansby which diagnostic functional imaging of the neural system, andespecially the peripheral nervous system, can be achieved in vivo.

Diagnostic imaging of nervous system function has a multitude ofpotential applications which will readily be apparent to the physicianor neurosurgeon and many of these are discussed further below. Thus forexample the possibilities would exist to visualize the impact ofneurofibrillary tangles as they develop, to locate and assess nervecompressions, to verify the effectiveness of surgical vagotomy and tomeasure the response of the injured spinal cord to attempts attreatment.

It has now been realized that particulate agents suitable for use ascontrast agents in diagnostic imaging modalities, especially MRI andPET, may be conjugated to nerve adhesion molecules and that followingadministration into body tissue, especially muscle, such agents areendocytosed by neurons having axon termini in that tissue and carriedalong the axons by axoplasmic flow thus allowing imaging of the axonsand of the nerves of which they form part.

The endocytosis of nerve adhesion molecule (NAM) labelled agents canalso clearly be utilized for the remote delivery of therapeutic agents,i.e. axoplasmic flow can serve to transport a therapeutically activeagent comprising a nerve adhesion moiety from its administration site intissue such as muscle to a remote site where it exerts itspharmacological effect. This is of particular interest where thesensitivity or accessibility of the remote site is such as to preventdirect administration of the pharmaceutical.

Thus viewed from one aspect the invention provides a method of treatmentof the living human or non-human (preferably mammalian) body to effect adesired therapeutic or prophylactic treatment or assist diagnosticinvestigation or surgical treatment thereof, said method comprisingadministering into a vascularized peripherally innervated tissue site(preferably a muscle although possibly also other tissue sitesinnervated by cranial, peripheral or autonomic nerves) or into othertissue sites innervated by a spinal root a particulate pharmaceuticalagent comprising a nerve adhesion moiety serving to promote neuronalendocytosis of said agent and a physiologically active or diagnosticmarker moiety capable of axonal transport from said tissue site, and,where said method is to assist diagnostic investigation or surgicaltreatment, detecting axonal transport within said living body of a saidagent having a diagnostic marker moiety, preferably by generating animage of at least part of said body.

Viewed from another aspect the invention provides the use of aparticulate pharmaceutical agent comprising a nerve adhesion moietyserving to promote neuronal endocytosis of said agent and aphysiologically active or diagnostic marker moiety capable of axonaltransport following neuronal endocytosis of said agent for thepreparation of a therapeutic, prophylactic or diagnostic composition foruse on administration into vascularized peripherally innervated tissueor into other tissue sites innervated by a spinal root in a method oftreatment of the living human or non-human body to effect a desiredtherapeutic or prophylactic treatment or assist diagnostic investigationor surgical treatment thereof.

Especially in the case of therapeutic or prophylactic Treatment, thepharmaceutical agent is preferably administered into a tissue site, suchas a muscle, having a volume of at least about ten times that of thegroup of nerve cells which are to transport the agent.

The term pharmaceutical agent is used herein Lo designate a substancecapable of exerting a desired therapeutic or prophylactic effect and/oracting as a tracer, label, contrast agent or other diagnostic markerdetectable in the intact living mammal. This substance may be a singlecompound but more generally will comprise a NAM coupled directly orindirectly to a physiologically active or diagnostically markedcompound. Diagnostic marking may for example be with radiolabels,chromophores, fluorophores, by virtue of magnetic properties, or withatoms or structures capable of higher or lower radiation (e.g. X-ray orsound) absorbance or reflectance than surrounding body tissue.Particulate NAM-coupled moieties may be coated or uncoated and if coatedthe coating may be selected to be broken down within the neuron afterendocytosis, either slowly or more rapidly, or to be maintained duringaxonal transport.

For the purposes of the present invention it should be appreciated thatwhile natural or synthetic, essentially inert, organic polymer particles(such as dextran coated microspheres or latex nanospheres) are capableof being endocytosed, these organic polymers unlike more specific andcomplicated molecules such as proteins, antibodies and antibodyfragments are not considered to be nerve adhesion molecules.

The mean particle size for the particulate pharmaceutical agents used inthe invention is conveniently in the range 5 to 100 nm, especially 8-70nm, more particularly 10 to 50 nm and preferably about 20-30 nm.

Many of the pharmaceutical agents that may be used in the method ofinvention are themselves novel and viewed from a further aspect theinvention provides a pharmaceutical agent comprising a nerve adhesionmolecule coupled (directly or indirectly) to an optionally-coated,particulate, physiologically active or diagnostically marked substance,with the proviso that for diagnostically marked substances the substanceis a metal oxide, metal sulphide or alloy.

For use to assist diagnosis, the pharmaceutical agent preferably has adiagnostic marker that can be detected non-invasively, e.g. by virtue ofits radiation emission or absorption characteristics or by virtue of itsmagnetic characteristics. For use in assisting surgery, for example toenable important nerve pathways passing through or near a wound site orother site undergoing surgical intervention, chromophores andfluorophores can also be used as diagnostic markers and in this instancein particular the use of non-particulate as well as of particulatepharmaceutical agents might be contemplated.

One especially important group of pharmaceutical agents for useaccording to the invention is that of NAM-coupled particulate inorganiccompounds, for example metal oxides, sulphides or alloys, where theinorganic material is selected for its magnetic properties, inparticular ferri- and ferromagnetism and more particularlysuperparamagnetism, or includes within an otherwise essentially inertmatrix atoms or molecules which are released gradually from the matrixto exert a therapeutic or prophylactic effect or which function asdiagnostic markers, e.g. radioisotopes or nuclides detectable upon MRspectroscopy. Many metal oxide structures may be utilized as theinorganic particles, and spinels and garnets have been found to beparticularly useful in this regard. It should however be stressed thatother well known inert and Preferably essentially water insoluble metalcompounds may be used, especially those having or capable of being dopedto exhibit cooperative magnetic properties and those having latticessuch as permit desired radioisotopes to be included. By alloys, mixedmetals are of course included. Organic particulate matrices may also beused to accommodate a therapeutic compound or a diagnostic marker.

As is clear from the above, this invention is especially concerned withimprovements in particulate pharmaceuticals which surprisingly result inProviding access to new patterns of distribution within the body whichwere never previously possible. As a result of these new patterns ofdistribution, a number of previously intractable problems in medicaldiagnosis and treatment have now been solved.

For the particulate pharmaceutical agents in particular, theimprovements include 1) improved control of particle size, 2)development of an effective means of affinity purification of theparticles, 3) demonstration of a means of filter sterilization of theconcentrated product late in the synthesis, and 4) widening the array ofelements and concentration of those elements applied to medical uses inmetal oxide or sulphide crystals or in alloys.

One of the most important consequences of these advances is thedevelopment of agents which can be delivered by, and make use of anentirely novel intraneural pharmaceutical route (IPR). In addition,these advances greatly simplify and reduce the cost of production ofrelated particulate agents with previously known uses.

In a preferred embodiment, the agent comprises particles with a coremetal oxide crystal, e.g. of spinel or garnet structure, coated forexample by dextran carbohydrate wherein the total size of the coatedparticle is between 100 and 500 Angstroms (10 to 50 nanometers) andwhere a targeting moiety (TM) is chemically bound to the coating in lowconcentration of TM per particle, preferably 1:1. The agent shouldpreferably be virtually free of particles lacking an active TM, andcompositions thereof are preferably sterilized by 0.2 or 0.1 micronmicrofiltration after final synthesis, affinity purification andconcentration.

The uses of a given version of the agent depend upon the elements andisotopes (nuclides) used in the initial precipitation step in which themetal oxide crystal core is precipitated and coated and also upon thetype of targeting moiety used. For each use, the nuclide and targetingmoiety may be selected to benefit both from the general advantages ofthe simplicities of the preparatory method and to take advantage of thenew types of pharmaceutical distribution which can be achieved bymaterials prepared in this way.

The inorganic particles in the preferred pharmaceutical agents usedaccording to the invention generally fall into one of five categories:

i) particles exhibiting cooperative magnetic properties, in particularsuperparamagnetism, e.g. ferrite particles such as inverse spinelferrites, and thus detectable by magnetic resonance or magnetometricmethods;

ii) particles incorporating a gamma or electron emitter radionuclide,and thus detectable by gamma detectors, scintigraphy or SPECT (singlephoton emission computed tomography) or thus capable of causingradiation treatment effects;

iii) particles incorporating an element of non-zero nuclear spin, e.g.scandium, capable of being detected by magnetic resonance spectrometry;

iv) particles incorporating a positron (β⁺) emitter radionuclide andthus capable of detection by PET (positron emission tomography)

v) particles incorporating a compound or element capable on release,e.g. during degradation of the particle, of effecting a desiredtherapeutic or prophylactic effect.

Metal oxide particles of the first two types are clearly known butscandium containing spinels or garnets and the particles of the lattertwo types are novel and in themselves form further aspects of thepresent invention.

Thus viewed from a further aspect the invention prove less aphysiologically tolerable particulate metal oxide, metal sulphide oralloy with incorporated therein a positron emitter radionuclide andpreferably an element having high positron affinity (e.g. higher thanthat of iron, for example lithium or zinc). Such high positron affinityelement containing particles are preferably spinels and are referred toherein as “spinel moderated positron emitters” (SMPE) These have severalunique and surprising qualities which enhance the image resolution ofPET.

Viewed from a further aspect the invention also provides aphysiologically tolerable particulate garnet or spinel with incorporatedtherein atoms of scandium, of a radioactive yttrium isotope, of a sixthperiod metal (e.g. a lanthanide), of a high MR receptivity nuclide (e.g.at least as high as ₇₁Lu¹⁷⁵), or of an element which on particledegradation has a desired therapeutic or prophylactic activity.

It will be appreciated that although the metals of the metal oxide,sulphide or alloy matrices of the particles of the invention may havenaturally occurring positron emitting isotopes the particles accordingto the invention have significantly higher than natural abundancecontents of these, e.g. for positron emitters an average of at leastone, perhaps 10 or more atoms per 100 nm crystal. The natural occurrenceof many β⁺ emitters is less than 1 in 10²⁰ and even one emitting atomper particle may suffice.

For other novel “doped” particles according to the invention, the activeor marker nuclei may be isotopes which occur naturally, e.g. asimpurities in naturally occurring oxides, sulphides or alloys—in thiscase again the particles according to the invention are distinguished bycontaining such atoms at higher than natural values, e.g. a hundred oreven more per 100 nm particle.

The particles of the invention may be coated or uncoated and may derivetheir physiological tolerability at least in part from such a coating.They may moreover be coupled to a biotargetting moiety, for example anantibody, an antibody fragment or another NAM.

The particles of types i) and ii) mentioned above are also preferably ofa spinel or garnet structure—the manufacture of particles of these typesis already well known and need not be described further here. By way ofinterest however it may be noted that superparamagnetic crystals of thistype have been proposed for use as MRI contrast agents in various patentpublications of Nycomed AS, Schering AG, Advanced Magnetics Inc, etc(e.g. U.S. Pat. No. 4,863,715 (Jacobsen) and U.S. Pat. No. 4,827,945(Groman)).

There are a wide variety of targeting moieties or NAMs which can be usedaccording to the invention. These include antibodies, monoclonalantibodies, antibody fragments, receptors, peptides such as endorphins,steroid molecules, viral fragments or coat proteins, cell surfaceantigens including various carbohydrates, lactins, immunoadhesins,neurotransmitter molecules, growth factors, and other proteins whichpromote endocytosis or the pharmaceutical agent by the axon termini. Theuse of lectins, such as WGA, is particularly preferred.

The synthesis of metal oxide crystals as particulates in stable aqueoussolution has been of interest in crystallography and in the paintpigment industry. However, many of the relevant advances have grown outof studies of magnetism.

Many of the agents described herein involve specially synthesizedversions of magnetite (Fe₃O₄). The crystal structure of magnetite isbased on a mineral called spinel MgAl₂O₄. However, when specificproportions of ferric and ferrous ions are used instead of magnesium andaluminum as the metal ions in the lattice: Fe(II)(Fe(III) )₂O₄, aparticular set of electronic alignments and exchanges are produced whichresult in spontaneous magnetization.

The basic structure of magnetite involves a close-packed, face centredcubic crystal of oxygen atoms with metal ions placed at interstitialspaces in the crystal (see FIG. 1). The interstices are divided into “A”sites and “B” sites which have different interstitial locations relativeto the oxygen array and which therefore give rise to two distinctsub-lattices within the crystal. In the naturally occurring mineral“spinel” (MgAl₂O₄) the A-sites are filled by Mg(II) and the B sites byAl(III). The assignment of atoms to sublattices is determined in part bysize. The A-sites allow atoms of 0.3 to 0.6 angstrom radius while theB-sites allow atoms of 0.6 to 1.0 angstroms. In a normal spinel crystal,the A-sites are filled by divalent atoms while the B-sites are filled bytrivalent atoms.

Magnetite is an “inverse spinel” crystal because it has trivalent ironin its A-sites, and a mix of divalent and trivalent iron in its B-sites.Each crystal subunit has 32 oxygens, 8 A-site Fe(III) atoms, 8 B-siteFe(III) atoms and 8 B-site Fe(II) atoms. The general formula for spinelferrites is Mt(II): (Fe(III))₂(O)₄, where Mt can be any divalenttransition metal or a charge balanced mix of monovalent and trivalentmetals of appropriate ionic radius.

The Fe(III) atoms in the A sublattice are positioned so as to oppose andcancel the spin magnetization of the Fe(III) in the B sublattice.However, after this cancellation, the 8 Fe(II) remaining in the Bsublattice have completely unopposed spin magnetizations. For each Fe₃O₄formula unit, there is a net magnetization of 4 Bohr Magnetons due tothe unopposed Fe(II) atoms. Each crystal subunit therefore has amagnetization of 32 Bohr Magnetons packed into a cube with a face thatis 837 pm in length.

The magnetization of a ferrite can be altered by substituting differentmetals into the various interstices. For instance, Mn(II) has amagnetization of 5 Bohr Magnetons, so creation of an inverse spinel withthe formula Mn(II)(Fe(III))₂O₄ should yield crystals with 5 BohrMagnetons per unit. The use of Zn(II) has a quite different effect. Ithas no unfilled d-orbitals and so has zero magnetic moment. However,zinc tends to enter A sites causing a normal spinel organization for thecrystal. Therefore, at each formula unit, a zero moment zinc opposes anFe(III) with a moment of 5 Bohr Magnetons resulting in a net moment of 5for the pair, the remaining Fe(III) are also unopposed, so the netmoment is 10 Bohr Magnetons per formula unit (80 Bohr Magnetons percrystal subunit).

In actuality, this situation can prevail only for a low percentage ofthe total number of sites in a larger crystal. Zn(II) is actually toolarge for the A sites (0.77 angstrom radius) so that as theconcentration of zinc exceeds 50%, there is a transformation intoinverse spinel structure. In this arrangement, Fe(III) opposes Fe(III)cancelling each other out, and the unopposed Zn(II) have no moment, sothe ferrite has a net magnetization of zero. This is sometimes useful inapplications such as the heteronuclear tracers described below in whichmagnetization is not necessarily desirable.

In 1955 the term superparamagnetism was proposed to describe thebehaviour of extremely small magnetic particles. The fundamental idea isthat there is sufficient thermal agitation in a small particle that thetendency for the magnetic dipole axis to flip into various orientationsis greater than the tendency to align as a coherent domain with a singlefixed axis.

As the particle size increases above a critical size in the range of 10⁶atoms, it becomes stable and coherently aligned as a spontaneouslymagnetized single domain. Below this critical size, the magneticsusceptibility is temperature and size dependent. Smaller particles athigher temperatures require stronger external fields to becomedetectably magnetized. Once magnetization is achieved, however, thetotal magnetization is related directly to the size of the particle.

The behaviour of a superparamagnetic particle is described by arelaxation rate which reflects the rate at which local magnetic momentswithin the particle will flip spontaneously. In order to flip, an energybarrier which is proportional to the volume of the particle and to theanisotropy of the material must be overcome. In a domain sized particle,the magnetization settles along one single axis because the energybarrier is too great to permit flipping at the temperature of theexperiment. At sub-domain size, the energy barrier is low enough thatthe flip rate becomes exceedingly rapid. The size at which thistransition occurs is temperature dependent and also dependent on thecomposition of the particle. (For present purposes the relevanttemperature for determining whether or not a substance issuperparamagnetic is body temperature).

By the substitution of some metals such as cobalt in place of some ofthe Fe(II) in the lattice, the crystals become more anisotropic and thistends to slow the rate or flipping and so lower the critical size for astable domain.

When larger ions are included in the crystal matrix, the spinelstructure cannot accommodate them. This is particularly important forthe use of elements from the lanthanide series. However, lanthanides maybe accommodated by the garnet crystal structure. The natural form ofthis crystal is Ca₃Al₂(SiO₄)₃ or 3CaO·Al₂O₃·3SiO₂. An analogousstructure is achieved with the composition Ln₃Fe₅O₁₂, wherein Ln is alanthanide element. (A common example made using Yttrium is called YIGor Yttrium-Iron-Garnet and is used for instance in lasers). Althoughsmall amounts of the lanthanides are accommodated within spinelcrystals, stoichiometries which favour garnet formation are moreimportant as larger percentages of lanthanides are included.

A novel type of spinel crystal developed and synthesized according tothe invention uses scandium in place of aluminum in the preparation ofcoated, colloidal spinel crystals. The most stable of these areMg(II)(Sc(III))₂O₄ or magnesium scandites. These are helpful vehicles inseveral of the applications described below. These crystals are notmagnetic. Scandium has stable trivalent chemistry but, unlike yttriumand lanthanides, is similar in ionic size to the remaining transitionmetals.

Methods for precipitating ferrites from metal salts date back into the1800's and several investigators have modified these methods in attemptsto develop improved ferrofluids. Elmore in Phys. Rev. 54: 309-310 (1938)explored ammonia precipitation of ultrafine ferrite particles in aqueoussolutions and first demonstrated that their aggregation increased whenthey approached an applied magnetic field.

A further step towards developing stable colloidal ferrofluids came in1965 with the development of a method for grinding magnetic materialsinto fine powders and then suspending them in oleic acid by sonication(see U.S. Pat. No. 3,215,572). Takada and Kiyama in Proc. Int. Conf.(ICF-1), U. Tokyo Press (Ed. Hoshino et al), p.69-71 (1970) reexplored avariety of methods for precipitating ultrafine crystals of magnetite anddeveloped a new oxidation method although this body of work did notaddress the problem of keeping the particles in suspension.

Reimers and Khalafalla in Bu Mines TPR 59:13 (1972) used an ammoniapeptization method to create aqueous suspensions or ground particles. Intheir initial method, an acid treatment followed by sonication is usedto induce interaction with solvent molecules to prevent clumping of theparticles and maintain suspension. Subsequently, they developed amodification of Elmore's ammonia precipitation method to create morestable, dilutable suspensions in which molecules of dodecanoic acid arechemically adsorbed onto the surface of the magnetite particle (seeKhalafalla and Reimers in IEEE Trans Mag 16: 178-183 (1980)). Thisyielded dilution-stable solutions of superparamagnetic particles.

Biologists became interested in small magnetic particles as-potentialmeans of carrying out biochemical separations and developed variousmeans of incorporating domain sized particles into beads. These did notneed to be soluble in the form initially used. However, building onmethods used to create dense immunospecific labels for electronmicroscopy, an aqueous technique developed by Molday (see U.S. Pat. No.4,452,773 and J. Immunol. Meth 52: 353-367 (1982)) opened the way to avariety of biological applications.

The Molday method involves an ammonia precipitation synthesis in whichdextrans are used to coat the magnetite. This results in an aqueoussuspension of superparamagnetic particles which can be conjugated to awide variety of types of molecules including antibodies and so used tocarry out various types of separations. The advantage of thesuperparamagnetism of the Molday particles is that they do not tend toaggregate magnetically unless they are in an applied magnetic field.This simplifies the preparation of more elaborate compounds whilepermitting recovering of the magnetic properties when they are wantedafter the synthesis is completed.

Whitehead et al (U.S. Pat. No. 4,554,088) developed a silane bindingtechnique in which clusters of superparamagnetic magnetite particleseach about 30 nm in size are bound in groups into larger particles about500 nm in diameter (now marketed as “AMI-25”). In the silane matrix, thesmall particles are held apart from each other and so retain theirsuperparamagnetism. They therefore do not aggregate and remainrelatively soluble. However, the total magnetic moment of the entirelarger particle is quite large so that biological separations can becarried out.

Sub-micron coated iron oxide particles have been proposed for use asintravascular X-ray contrast agents and a number of other medical useshave been described for other superparamagnetic particles includingmagnetic confinement for blockage of fistulas and thrombosis ofaneurysms, use in producing focal diathermy for treatment of infection,selective removal of tumour cells from bone marrow, and use as MRIcontrast agents.

There have now been a variety of clinical studies in which MRI contrastis achieved by intravenous injection of ferrites for evaluation of liverand spleen tumours and also after oral intake as a gastrointestinalcontrast agent. In these cases, it is the particulate nature of thematerial that is used to achieve useful distributions in the body,either by their uptake by reticuloendothelial cells in liver and spleen,or by their confinement to the GI tract because of their indigestiblenature.

Paramagnetic contrast agents such as gadolinium-DTPA act primarily byaltering T₁ relaxation rates. Superparamagnetic agents cause their MRIcontrast enhancing effect in a rather different fashion. When theexternal main MR field is applied, the particles are organized intoacting as powerful microscopic magnets scattered through the tissuebeing imaged. These particles therefore result in large numbers of localinhomogeneities in the larger field to which the protons are exposed. Inthe vicinity of an activated magnetite particle, therefore, the Larmorfrequency of the protons is shifted away from resonance with the RFpulse (away from 200 MHz in a 4.7 Tesla field) and so generate a lessintense signal. At larger distances from a magnetite particle, thecontrast agent's field will cause smaller changes in Larmor frequency,so that although the RF pulse is still fully absorbed, the slightdifferences will accelerate dephasing of the protons (i.e. shorten T₂).This is similar to the effect caused by the local field inhomogeneitiesin the main magnet. However, because the magnetite particles arethemselves tumbling and moving over relevant time scales in the signalcollection sequence, the rephasing pulses are ineffective. Therefore,particularly in T₂ weighted images, a single magnetite particle can havetremendous impact.

Indeed, experiments conducted in our laboratory show that a single 40 nmparticle of magnetite can drive T₂ to less than 10 milliseconds in anarea more than 10 microns in diameter. This is why exceedingly lowconcentrations of magnetite particles in the range of 50 picomoles/litercan be effective. Even greater sensitivity may be achieved by usingspecially designed pulse sequences based on current gradient echotechniques which are particularly sensitive to local variations inmagnetic field homogeneity.

Widder (U.S. Pat. No. 4,849,210) and Jacobsen (U.S. Pat. No. 4,863,715)demonstrated the effectiveness of suspensions of ferromagnetic particlesas intravenous MRI contrast agents with various methods of synthesis.Groman (U.S. Pat. No. 4,827,945) provided a number of additional methodsof synthesis of superparamagnetic particles and suggested the MRintravascular use of a wide range of labelled particles analogous tothose disclosed for in vitro use by Molday. Although the compounds theydescribe are physically very similar to those disclosed by Molday (U.S.Pat. No. 4,452,773) they discuss sterilization techniques and methods ofuse involving diagnostic MRI.

However, the particles produced by the methods of Groman vary in sizefrom 100 to 5,000 Angstroms, cannot be filter sterilized in concentratedfinal form, and cannot be effectively purified by affinitychromatography since, like the compounds of Molday, they contain manyconstituents which will not pass readily through agarose based affinitymedia late in the preparation. Because of the need for autoclaving ofthe Groman products, the use of delicate protein ligands is severelylimited because they cannot withstand autoclaving. It is possible tocarry out the synthesis of Groman using ultraclean facilities so thatfinal sterilization of the product is less important but this addsconsiderably to the expense of manufacture.

The products of the Groman synthesis are not useful for radionuclideimaging of axonal transport because most of the particles are too largeto be endocytosed by neurons and only a small proportion will carryactive targeting moiety thus leading to unnecessarily large doses ofradiation to achieve the required intraneural dose and leading tounnecessarily high tissue radiation background levels. Similarly, forMRI applications, the product of the Groman synthesis requiresunnecessarily large injections into muscle to achieve the needed dose ofsmall, specifically labelled sterilized particles desired.

The current invention achieves particular improved characteristicsthrough the discovery that the use of repeated purification steps duringthe synthesis greatly improves the performance of the particles asbiochemical reagents. These purifications remove dissolved metal ions asthey appear during the synthesis since they can precipitate as hydrousoxides which impair the gel flow characteristics of the preparationduring the synthesis. In addition, by using serial filtration stepsafter the initial precipitation, particles may be selected which areless than 500 angstroms in size (including the dextran coat). This helpsassure the flow characteristics of the particles through the remainderof the synthesis and results in the production of only 100-500 angstromparticles which have a number of physiological advantages but are alllarge enough to retain superparamagnetic function.

Finally, and most importantly, when all these measures are taken, it ispossible to take advantage of the versatility and convenience ofre-usable agarose based affinity chromatography media to remove allparticles which are not bound to a targeting moiety as well aspermitting the discard of all particles whose bound targeting moiety hasbeen inactivated or otherwise lost its specificity during the syntheticprocess. The potential to use these media is quite important since thispermits the preparation of affinity media with a wide variety of ligandswhich can be used to purify a correspondingly wide variety of targetedparticles. Although there are many applications of large (10 to 40micron) magnetic beads as supports for affinity separations of otherkinds of (non-magnetic) molecules and cells no previous preparation hasachieved the affinity purification of the small superparamagneticparticles themselves upon a standard affinity chromatography matrix.

The final result is an agent with very nearly one active targetingmoiety per particle with all particles selectively active and smallenough for effective use. This can then be concentrated or formulated asdesired and filter sterilized in small volume if necessary. The finalsterilization can be with conventional 0.2 micron filters for bacterialclearance or with 0.1 micron filters to assure removal of smallmycobacterial contaminants.

An alternative method of obtaining high specific activity is to actuallycoat all of the particles in the preparation with a large number ofmolecules of the targeting moiety. This has the undesirable effects ofgreatly increasing the expense of the product when the targeting moietyis expensive to produce, increasing the antigenicity of the particle,and in many cases, altering the distribution of the particle inundesirable ways. It is well known from work in affinity chromatographyon solid supports that spacing and density of affinity ligands arecrucial determinants of efficacy.

There has been considerable interest in the medical uses of varioustypes of microspheres and nanospheres. The composition of such particlesinclude latex polymers from various methacrylates, polylactic acid,protein/ albumin, lipids and various other materials (see for exampleProc. Soc. Exp. Biol. Med 58: 141-146 (1978), AJR 149: 839-843 (1987),J. Cell Biol. 64: 75-88 (1975), J. Microencaps 5: 147-157 (1988), andRadiol. 163: 255-258 (1987)). These particles have been used as drugdelivery systems, imaging agents, and for histological studies of axonaltransport. They offer unique patterns of metabolism and biodistributionand continue to be the subject of intense investigation by many groups.The uses of such particles for in vivo diagnostic imaging of axonaltransport or for the delivery of large numbers of atoms forheteronuclear imaging are among the new uses for microspheres describedherein. The use of such particles as part of a drug delivery system thatemploys an intraneural route and axonal transport is also described herefor the first time.

As mentioned above, the particles and the particulate agents of theinvention preferably comprise therapeutically or prophylactically loadedor diagnostically marked inorganic crystals, e.g. β⁺ emitter markedmetal oxides.

Currently, the principal uses of positron emission tomography are insituations in which relatively short half-life emitters such as ₆C¹¹(t^(½)=20.3 minutes, 0.960 MeV), ₇N¹³ (t^(½)=9.96 minutes, 1.190 MeV),₈O¹⁵ (t^(½)=2.03 minutes, 1.723 MeV), and ₉F¹⁸ (t^(½)=109.7 minutes,0.635 MeV) are effective. However, for diagnostic or treatmentsituations such as the use of monoclonal anti-tumour antibodies or forimaging of axonal transport, it is sometimes necessary to allow severaldays for adequate tissue distributions to be achieved. There are avariety of relatively long half-life positron emitting nuclides,however, all of these have previously proven to be difficult to keepfirmly bound to proteins over the necessary two to three days.

As demonstrated in FIG. 2, there are a number of nuclides emittingpositron and electron β-particles all of which can be included in metaloxides, e.g. spinels such as ferrites, either as substituents in thecrystal lattice or as seeds, e.g. ZrO₂, inside ferrite spinel shells.This provides a new and unique way of delivering these various nuclidesto various medically useful locations in the body in a wide variety ofnew concentrations and half lives. A single ferrite particle can be usedto attach hundreds or thousands of β-emitting atoms to a singleantibody, thus far exceeding the intensity of signal per antibodymolecule available in current preparations which generally provide oneemitting atom per antibody molecule.

The uses for these various beta emitting ferrites are protean andinclude imaging tasks as well as a number of treatment modalities. Fromone point of view, this array of possible nuclide preparations presentsa wide range of half lives and particle energies which can be used forvarious tasks. The great simplification provided is that all of thesecan be manufactured and delivered by means of essentially similarmolecules with effectively identical chemistry. Most of these nuclidesdecay to daughters which are also easily accommodated in the crystal andtherefore do not involve loss of integrity of the particle as decayprogresses.

In one set of embodiments, the positron emitting isotopes of manganese(₂₅Mn⁵²), iron (₂₆Fe⁵²), cobalt (₂₇Co⁵⁵), or rhodium (₄₅Rh⁹⁹) are usedin the synthesis of spinel particles, e.g. sub-domain sized,superparamagnetic ferrite particles. The inclusion of cobalt ormanganese in this type of ferrite has previously been difficult toachieve efficiently, but it is possible to reliably introduce cobalt,manganese, or other metals in amounts up to ⅓ of the number of metalatoms per formula unit, e.g. with the remaining ⅔ being Fe(III) if thestoichiometry of the desired crystal structure, e.g. garnet or spinel,is carefully considered and factors such as pH, temperature, andprecursor metal salt and coating compound concentrations and theduration of heat incubation after precipitation are carefullycontrolled, preferably after optimization by routine experimentation.Thus as an example, for dextran coated particles it has generally beenfound advantageous to precipitate out from a saturated dextran solution.Thus all the divalent metal atoms may be replaced as opposed to the ½ orfewer suggested by Groman in U.S. Pat. No. 4,827,945.

These particles may be synthesized in such a way that they are stablycoated with dextran or other hydrophlic molecules and the coating maythen be activated and bound covalently to antibodies or any type ofnerve adhesion molecule. Particles so fashioned will be detectable uponPositron Emission Tomography (PET) as positron sources, and also uponMagnetic Resonance Imaging (MRI) as superparamagnetic particles. Some ofthese will also be detectable upon Magnetic Resonance Spectroscopy (MRS)as high receptivity nuclei at selected frequencies or on X-ray CTscanning where the Z-number and particle concentration is sufficient.

In positron ferrites made with ₂₅Mn⁵² the emission detection is based onthe 0.511 MeV annihilation photons due to positron decay (β+27.9%, 0.575MeV, E.C. 72.1%) with a half life of 5.59 days and associated gammaemissions of (100%, 1.434 MeV; 94.5%, 0.935 MeV; 90%, 0.744 MeV; 5%,1.33 MeV; 4%, 1.25 MeV; 3%, 0.85 MeV) to ₂₄Cr⁵² which is stable. This isa decay half life which is quite well suited to long nerve transportsand to full monoclonal antibody distribution for tumour studies.Further, with a relatively low positron energy of just 0.575 MeV, thespatial resolution is substantially better than any positron emitter inactive clinical use including ₉F¹⁸. The high gamma emission may make₂₅Mn⁵² less attractive for clinical use in some situations, but asindicated by FIG. 2, there are many alternatives.

Positron ferrites can also be made with ₂₆Fe⁵² which undergoes positrondecay (β+56%, 0.804 MeV; EC 43.5%) with a half life of 8.275 hours andassociated gamma emissions (99.2%, 0.169 MeV) to ₂₅Mn^(52m) which ismetastable and decays with a half life of 21.1 minutes by positron decay(β+96.27%, 2.631 MeV; EC 1.53%) and associated gamma emission (97.8%,1.434 MeV) to stable ₂₄Cr⁵² as well as by isomeric internal conversion(2.2%, 0.378 MeV) to ₂₅Mn⁵².

This type of positron ferrite has the advantage of a strong positronemission signal during the day of injection with a fairly rapid declinetowards the continuing positron emission of the ₂₅Mn⁵² with a 5.7 dayhalf life. This is particularly useful in neuropathy studies where aninitial assessment of rate of transport is desired with a follow-upstudy done at several days to assess the amount of transport. At thetime of the initial study, only a small fraction of the intramusculardose will have entered the nerve, so a relatively high activityinjection is needed. However, after several days, the amount in thenerve will be much larger, and it is then helpful to minimize thecontinuing absorbed dose to the patient by delivering it as the 2.2% ofthe ₂₅Mn^(52m) converted to ₂₅Mn⁵².

An intermediate half life can be provided by positron ferrites made with₂₇Co⁵⁵ which undergoes positron decay (β+77%, 1.54 MeV; EC 23%) with ahalf life of 17.5 hours and associated gamma emissions (75%, 0.93 MeV;16.5%, 1.41 MeV; 20.3%, 0.477 MeV; 7%, 1.32 MeV; 3%, 1.37 MeV) to₂₆Fe⁵⁵. This nuclide of iron then decays slowly by K-shell electroncapture (0.006 MeV) with a half life of 2.7 years to ₂₅Mn⁵⁵ which isstable.

Although the half life of this cobalt positron emitter may be useful forsome studies, its use is inhibited by the decay pattern of ₂₆Fe⁵⁵; theenergy of the photon is quite low, but the irradiation continues for along time and virtually all the energy is deposited within tissue asnon-penetrating radiation. This type of ferrite, however, does have theadvantage of yielding a ferrous ferrite which is a chemically quitestable metal oxide that is cleared from the body differently than ioniciron. Further, unlike positron ferrites decaying towards an increasingcomposition of chromium or titanium, these compositions result inchemically stable ferrite particles with good magnetic properties and soremain effective superparamagnetic MR contrast agents as decayprogresses.

A fourth type of positron ferrite can be synthesized with ₄₅Rh⁹⁹ whichundergoes positron decay (1.03 MeV) with a half-life of 16.0 days and noassociated gamma emission to ₄₄Ru⁹⁹ which is stable. This is a longerhalf life than will generally be needed but may be helpful intransneuronal transport studies intended to cross a synapse fortransport in a second nerve in a chain. In particular, this could behelpful in studies of spinal cord injury. Also this sort of positronferrite could be used in studies intended to assess the acute effect ofsurgery, where a diagnostic study is done and then a second study isrequired several days after the surgery to assess whether anaccumulation of transported molecules at a compression site hadcommenced to clear.

The decay for ₂₁Sc⁴³ (β+78%, 1.22 MeV; EC 22%) and associated gammaemission (22%, 0.373 MeV) with half life of 3.9 hours to stable ₂₀Ca⁴³make this very promising for clinical work. Particularly for rate oftransport studies in the lower extremity which are carried out after afew hours, this may be a completely adequate half life to allowobservation of the advancing front of the transport pulse. Thesubstantial increase in ionic radius and the tendency to change fromtrivalence to divalence upon transition from Sc to Ca will be disruptiveto the spinel crystal, but this may aid in the more rapid metabolism ofthe particles.

Except for calcium, all of these nuclides are accommodated in the spinelferrite crystal, although the chromium decay products from ₂₅Mn⁵² and₂₆Fe⁵² will generate some regions of spinel chromite (FeCr₂O₄) withinthe inverse spinel ferrite (Mt[II]O:Fe[(III]₂O₃) crystal. Similarly,some regions of ilmenite, perovskite, and titanium spinel will form inconsequence of e.g. ₂₃V¹⁸ decay. In any case, the transitions due tonuclear decay will not affect the biodistribution of the tracers on thetime scale of the imaging studies. The particles degrade slowly afterinitial concentration in the reticuloendothelial system of liver, lungs,and spleen.

The optimal method for producing ₂₆Fe⁵² with minimal ₂₆Fe⁵⁵contamination is by the irradiation of ₂₄Cr⁵⁰ enriched chromium withcyclotron generated 38MeV ₂He⁴ beams (₂₄Cr⁵⁰(α, 2n)₂₆Fe⁵²) withsubsequent acid extraction, oxidation, evaporative drying, ether phaseseparation, redrying and filtration for sterilization (see Zweit Int. J.Radiat. Appl. Instrum. Part A, Appl. Radiat, Isol 39: 1197-1201 (1988)).Other reactions available for the production of ₂₆Fe⁵² include ₂₅Mn⁵⁵(p,4n)₂₆Fe⁵², ₂₄Cr^(nat)(α, xn)₂₆Fe⁵², ₂₄Cr^(nat)(₂He³, xn)₂₆Fe⁵²,₂₈Ni^(nat)(p, spall)₂₆Fe⁵² with subsequent acid extraction andpurification by anion exchange chromatography, wherein ₂₄Cr^(nat)includes ₂₄Cr⁵⁰ (4.35%), ₂₄Cr⁵² (83.79%), ₂₄Cr⁵³ (9.50%), and ₂₄Cr⁵⁴(2.36%).

₂₅Mn⁵² may also be synthesized by standard techniques including ₂He³activation of Vanadium ₂₃V⁵¹(₂He³, 2n)₂₅Mn⁵² (see Sastri Int. J. Appl.Rad. Isol. 32: 246-247 (1981)) or other cylcotron reactions including₂₄Cr⁵²(p,n) ₂₅Mn⁵², ₂₄Cr⁵²(d,2n)₂₅Mn⁵². Methods for ₂₇Co⁵⁵ include₂₆Fe⁵⁴(d,n)₂₇Co⁵⁵, ₂₆Fe⁵⁶(p,2n)₂₇Co⁵⁵, ₂₆Fe^(nat)(₂He³,xnp)₂₇Co⁵⁵,₂₅Mn⁵⁵(₂He³, 3n)₂₇Co⁵⁵, ₂₅Mn⁵⁵(α,4n)₂₇Co⁵⁵, wherein ₂₆Fe^(nat) iscomposed of ₂₆Fe⁵⁴(5.82%), ₂₆Fe⁵⁶(91.8%), ₂₆Fe⁵⁷(2.1%), and₂₆Fe⁵⁸(0.28%).

Generator techniques in which a longer half-life parent nuclide issynthesized and transported to the clinical site with subsequentextraction of the clinically useful daughter nuclide just prior to usecan be arranged for several useful metals. These include ₄₆Pd¹⁰⁰ (4.0dK,γ)→₄₅Rh¹⁰⁰ (20h β+), ₇₄W¹⁸⁸ (69d β−: 188 m,18 m γ) →₇₅Re¹⁸⁸ (16.7β−),and ₇₆Os¹⁹⁴ (6.0y β−)→₇₇Ir¹⁹⁴ (17.4h β−).

A proposed cyclotron ₂₁Sc⁴³ synthesis involves the following schemewhich would apply for alpha particle bombardment of ₂₀Ca⁴⁰ (thermalneutron cross section=0.43 barns):

₂₀Ca⁴⁰(α, n) ₂₂Ti⁴³→β+(0.56 s)→₂₁Sc⁴³

₂₀Ca⁴⁰(α, p) ₂₁Sc⁴³

₂₀Ca⁴⁰(α, d) ₂₁Sc⁴²→β+(0.68s)→₂₀Ca⁴²

₂₀Ca⁴⁰(α,2n) ₂₂Ti⁴²→β+(0.2s)→₂₁Sc⁴²→β+(0.68s)→₂₀Ca⁴²

₂₀Ca⁴⁰(α,xn) ₂₂Ti^(x)

₂₀Ca⁴⁰(α,3n) ₂₂Ti⁴¹→β+(0.69s)→₂₁Sc⁴¹→β+(0.60s)→₂₀Ca⁴¹

₂₀Ca⁴⁰(α,4n) [₂₂Ti⁴⁰]

The calcium and scandium are readily separated either by phaseseparation (see Hara in Int. J. Appl. Rad. 24: 373-376 (1973)) or bychromatography (see Kuroda in J. Chrom 22: 143-148 (1966)) which alsopermits separation of any titanium.

These and other transition metal or lanthanide nuclides can be used inthe synthesis of radioactive metal compounds (e.g. a metal oxide, metalsulphide or alloy, such as a ferrite) for use in monoclonal antibodybased treatment of tumours by irradiation. Here again, thebiodistribution and clearance of the delivered radionuclides is quitedifferent from single atoms chelated to the proteins. Intravascularinjection of Fe⁵⁹ labelled particles of the type described demonstrateda biphasic plasma half-life with about ¾ of the dose being cleared tospleen, liver, marrow, and slightly to lung over 1-2 hours, but with asubstantial fraction of the dose demonstrating a quite prolonged plasmahalf life of many hours. Each antibody molecule can be used to deliverseveral hundred or several thousand atoms of the desired nuclide soachieving a high local dose. It should also be noted that bindingmultiple emitter atoms to a single protein molecule has been known torapidly destroy the protein—this problem is substantially alleviated bythe SMPE particles because the emitting nuclei are up to 100 angstromsdistant from the NAM—thus the chance of any electron, positron, orgamma-ray interacting with the targeting NAM is reduced by severalorders of magnitude. Methods developed for antibody delivery of ₃₉Y⁹⁰can be applied with a far higher concentration of this nuclide includedin a conjugated ferrite. Another treatment problem where theseβ-emitting ferrites could be useful is in improving the current methodsof intra-articular radiotherapy in rheumatoid conditions.

Another means of delivery for β-emitting ferrites is by preparingsuspensions of particles in the pre-mix of various tissue glues. After asurgical resection of a tumour, particularly when near an eloquent areaof brain, it is often necessary to leave a thin shell of tumour behindon the brain surface. Common practice currently involves the use ofvarious tissue glues to attach a number of radioactive seeds to theresidual tumour surface. This method is tedious, leaves no means forremoval of the metal without repeat surgery, and causes artefact onfuture CT and MR scans which makes it difficult to assess the results oftherapy. If a colloidal solution of β-emitting ferrite is prepared in atissue glue component, this can be applied rapidly to the tumoursurface, minimizing exposure to the surgeon and operating staff. Theparticles are biodegradable, so will be resorbed over weeks. This methodalso permits the use of a variety of different nuclides depending on theenergy, penetration, or half-life desired.

Thus viewed from a still further aspect the invention provides acomposition comprising a cell adhesion moiety-coupled radionuclide and atissue glue.

The tissue glue may for example be based on a clottable protein such asfibrinogen; thus for example a glue such as “TISSEEL” from ImmunoDanmark A/S of Copenhagen) may be used. In two part systems such as thisthe NAM-conjugated particles are preferably in the protein containingcomponent.

Magnetic properties of the β-emitting vehicle can also be used to helpcontrol delivery. This method can be used with or without conjugationwith antibodies, and employs the selective catheterization techniques ofinterventional radiology. An arterial catheter can be introduced nearthe tumour, or ideally, at a tumour feeding vessel. A magnetic field canbe applied by means of multiple external current rings so as to bestrongest in the vicinity of the tumour. This can be achieved with themagnetic stereotaxy device described in U.S. Pat. No. 4,869,247. Finallya venous catheter system which is itself strongly magnetized andfurnished with a magnetized intravascular filter is introduceddownstream of the tumour in one or several draining veins or centrallyin the atrium. A highly energetic β-emitting ferrite is slowly injectedvia the arterial catheter. The progress of the particles through thetumour is slowed by the external field and by any antibodies which haveaffinity for tumour antigens detectable in the vasculature. Afterpassage through the tumour, the particles are collected on the venousmagnetic catheter/filter and so can be removed without exposing theremainder of the body to the radiation. If a positron emitter is used,it is possible to verify the effectiveness of the control of the speedof the ferrite, and if the filtration is effective, then a second stagetreatment can be done with ferrite particles including highly toxicalpha emitting nuclides such as ₇₈Pt¹⁸⁶ (K, α 4.23 MeV, 3h). Particlescan also be heated with tuned microwave irradiation during their transitfor a synergistic diathermy effect.

Turning now to PET image resolution, one of the limitations on scanningresolution is a result of the distance travelled by the positron afterthe decay event but before electron-positron annihilation. This distanceis dependent upon the energy of the characteristic β emission for agiven nuclide. The maximum range for an ₉F¹⁸ positron emitted at 0.64MeV is 2.6 mm while the particles from ₃₇Rb⁸² decay emitted at 3.35 MeVtravel up to 16.5 mm before annihilation. Along this path (see FIG. 3),the positron loses energy by interacting with the electrons of atoms itpasses, causing a variety of ionizations and excitations. Only when mostof the kinetic energy is expended does the positron interact with anelectron in a matter-antimatter annihilation reaction generating two0.511 MeV photons travelling approximately 180° away from each other.The residual momentum of the positron at the time of the annihilationimparts some translational momentum to the emitted photons resulting inan angle between the two which differs from 180°. Measurements of thisangle reflect the nuclide and the medium in which the energy losses andsubsequent annihilation take place.

It has been known for some time that the distance of travel of thepositron prior to annihilation is proportional to the density of themedium. The density of magnetite is 5,180 kg/m³, just over five timesgreater than most animal tissues and, according to classicalcalculations based on electron range measurements, this potentiallyresults in an 80% decrease in the maximum distance travelled by apositron travelling in magnetite as opposed to travelling in tissue.There is an increase in Brehmsstrahlung braking radiation proportionalto the effective Z number of magnetite (which=52), but this onlyaccounts for 1% of energy loss for a population of positrons.

The numbers stated above for travel of the positron before annihilationreflect maxima. In fact during positron emission, the decay energy isdivided between the positron and a neutrino and the division isvariable, thus resulting in a population of energies.

The mean energy of a positron from a given nuclide is about ⅓ of themaximum usually given as the particle energy. The means positron energyfrom ₂₅Mn⁵² is 0.19 MeV and in magnetite this classically would resultin a range of about 20 microns if the travel were entirely in magnetite.

However various elements have characteristic positron affinities andthese have profound impact on positron lifetimes. Therefore, theclassical view of positron range in relation to a general densitymeasurement proves to be a substantial oversimplification.

The positron affinities of a variety of nuclides are included in FIG. 2.It can be seen that by using high affinity nuclides such as lithium inthe β-emitter loaded particles, the positron range can be furtherdecreased.

In addition, it has been learned that defects in a crystal can causetrapping of positrons. Defects in YBaCuO_(x) perovskite crystals areparticularly effective at positron trapping even when these materialsare not in a superconducting state, however, even mechanical stressdefects in metals are fairly effective. There are also effects due tothe magnetic field generated by a moving positron and its interactionwith the spontaneous field of a material such as magnetite, as well aselectron interaction enhancement effects due to the number of unpaired,anti-spin matched electrons from d or f orbitals in the particularspinel used for the particulate shield.

The consequence of these considerations is that it is possible to beginwith a crystal seed of a positron emitting nuclide including severalthousands atoms of the emitter and then to precipitate a lithium or zincdoped, defected, magnetite shield around the positron emitting core.This shield will cause a very large fraction of the emitted positrons toundergo all of their ionization producing collisional losses within theparticle and therefore to annihilate without ever leaving the particle.Those positrons that do emerge from the surface of the particle withoutbeing affected by reflection or surface trapping effects will have agreatly reduced energy distribution, travel far shorter distancesthrough tissue, and create far fewer ionizations in tissue per decayevent than standard unshielded positron emitters.

The annihilation photons themselves are relatively unaffected by thepresence of ferrite as opposed to tissue in their surroundings.Therefore, there will be a very large decrease in tissue ionizationswith only a trivial decrease in photon emissions. Further, the photonemissions will all take place far closer to the location of the actualtracer atom, typically within several microns rather than withinmillimetres and this will result in an improvement in the spatialresolution of the PET scan. Further, the annihilations, as a population,will have lower momentum and this will shift the population annihilationangle closer to 180°, further improving the resolution of the scan.

Where β-particles are used for treatment rather than primarily forimaging, this shielding can be used to achieve extremely limited rangesof cytotoxic ionization injury.

The range of the emitted β-particles can be designed to be not muchgreater than the size of a single target cell, thus limiting effectiveirradiation to only those cells that actually ingest the particle andtaking advantage of the terminal Bragg peak effect which increases theionization rate for a low energy positron just before annihilation. Ashort half life emitter could be used to minimize the effect ofincreasing exposure range with digestion of the coating (which may takedays) and multiple treatments could then be carried out. Largerparticles can be used without magnetic aggregation by composing theshell of less magnetic nuclides.

A quite different set of particles, e.g. mixed spinels, may be used forspectroscopic tracing and heteronuclear imaging methods. When largepercentages of ₃Li, ₂₁Sc, ₂₇Co, ₂₅Mn, ₂₉Cu, ₅₉Pr, ₇₁Lu, or ₇₅Re areintroduced into ferrite crystals these become vehicles for deliveringlarge groups of those atoms to a desired site. These elements and theirvarious isotopes have high nuclear resonant receptivity when in theappropriate oxidation state and electron/chemical environment and so theMR machine can be used as a spectrometer to detect the presence of thesecrystals. Table I lists a series of nuclei with relatively highreceptivity. Any high receptivity metal in an oxidation state whereelectrons do not produce confounding relaxation (e.g. Mn⁷⁺, Co³⁺) or inwhich d-electron orbitals are entirely empty (Sc³⁺) or full. (Zn²⁺) are.particularly amenable. The chemical environment is also important tominimize the effects of quadrupolar relaxation for nuclei with I>½.

Nuclei such as F¹⁹ and In¹¹⁵ can be included in compounds which can thenbe included or embedded in microspheres of latex, protein, polylacticacid or other polymers and these can then be introduced into the axon insufficient quantity to achieve F¹⁹ or In¹¹⁵ imaging. F¹⁹ is also quitesuitable for labelling a variety of small molecules which aresusceptible to effective axonal transport. Compounds incorporating suchnuclei may also be included in the coating of metal compound particleswith a targeting moiety also present in the coating.

One optimal method in this regard is to use individual chelated scandiumatoms where the chelate is conjugated to a small nerve adhesionmolecule. By using a very small carrier, it can be assured that thetumbling rate of the scandium atoms is high enough to permit standard MRdetection. Where particles are used, the breakdown of the particleinside the neuron will slowly release scandium ions which will becomeimageable as they are freed from the particle and so begin to tumblerapidly.

Because of the very great abundance of Na²³, imaging with this nucleusto create a generally useful anatomical image of the patient is readilyachieved. Superparamagnetic particles such as ferrous ferrites areeffective relaxation agents for sodium and so can be used as axonallytransported contrast agent to study nerves upon sodium imaging.

TABLE I Nuclides with usefully large MR receptivity and theircorrespolnding frequency for 4.7 T MRS. Nuclei in italics (H, F, Na, P)are commonly used in MR spectrospcopy but are not readily included inmetal oxide particles. Frequency in MHz Nuclide MR receptivity at 4.7 T₁H¹ 1.000 200.0 ₃Li⁷ .270 77.6 ₉F¹⁹ .830 188.2 ₁₁Na²³ .093 53.0 ₁₅P³¹.066 81.0 ₂₁Sc⁴⁵ .301 48.6 ₂₃V⁵¹ .381 52.6 ₂₅Mn⁵⁵ .175 49.4 ₂₇Co⁵⁹ .27747.2 ₂₉Cu⁶³ .064 53.0 ₄₁Nb⁹³ .482 48.8 ₄₉In¹¹⁵ .332 43.8 ₅₃I¹²⁷ .09340.0 ₅₉Pr¹⁴¹ .260 54.0 ₇₁Lu¹⁷⁵ .048 22.6 ₇₅Re¹⁸⁷ .086 45.6 ₈₁Tl²⁰⁵ .140115.4 ₈₃Bi²⁰⁹ .137 32.2

Using a double tuned coil or multiple coil MR system, a high gradientproton image may be made and a selected voxel may then be evaluated atthe appropriate MR observation frequency. The presence of the givennucleus with the appropriate spectral appearance confirms the presenceof the tracer and also makes quantitation possible.

The sensitivity of these various nuclei for NMR is sufficiently greatthat actual scandium, or other tracer, imaging can be carried out whendelivered quantities are sufficient. This produces a positive imageroughly similar in appearance to those resulting from some currentnuclear medicine imaging studies. These uses of these nuclides are alsoapplicable to several of their isotopes, both stable and radioactivewith some variation in gyromagnetic ratio for the various nuclides.These variations also can provide multiple additional frequencyselectable tracers for spectroscopy or heteronuclear imaging.

Using particle types and delivery targeting systems as described above,a different group of metals can be used instead of the β-emitters toachieve the very short range radiotherapy effect. These are a variety ofnuclides in which decay is by K-shell capture. Although decay in thesenuclides involves collapse of an electron into the nucleus, theresulting vacancy causes effects among the remaining electrons whichresult in Auger and Coster-Kronig electron emissions. These haveextremely low energies and resulting ranges of micron and sub-microndistances, although several such electrons may be emitted for eachsingle decay event. An optimal nuclide with this behaviour is ₄₆Pd¹⁰³which is a pure K-capture nuclide with a 17 day half life; ₂₄Cr⁵¹ mayalso advantageously be used.

By analogy with the multiple tracer methods described above for MRspectroscopic nuclides, it is also possible to use various transition orlanthanide metal radionuclides to prepare multiple metal conjugatedantibody tracers with the intent of providing them with characteristicgamma emission signatures. Here, the positron emission or MR contrasteffect could be used for localization and then the gamma emissions couldbe evaluated for energy level/frequency. In this fashion, multipledifferent gamma labels could be distinguished as a means for image basedtumour diagnosis by multiple antibody labels.

The particles used generally should be metal compounds capable ofprecipitation to a stable colloid having a particle size suitable forcell uptake and having a surface capable of being coated with or boundto biochemically useful materials, e.g. carbohydrates or proteins.

As a dense material, ferrite particles are effective X-ray contrastagents. By substituting high Z metals (e.g. elements of atomic number 50and above, especially sixth period elements) into the lattice, theireffectiveness can be further enhanced and the necessary dose thusdecreased. This is illustrated by FIG. 4 hereto which shows a CT imageof a phantom with wells containing similar concentrations of Mg/Tb andFe/Fe particles showing the greater X-ray opacity of the former. Wells11 to 17 contained the following X-ray contrast media:

Concentration Well No. Material (mg/ml) Field Units 11 Mg/Tb (III)  30*411 12 Mg/Tb (III)  10* 150 13 Fe/Fe (III)  30* 101 14 Fe/Fe (III)  10*0 15 Metrizamide  33 250 16 Air — −1017 17 Metrizamide 100 447*Concentration of the trivalent metal

This sort of technique is particularly useful for axon transport imagingtechniques and the evaluation of spinal root compression by herniateddisks by CT scanning. Since higher particle concentrations are neededfor CT than for MRI, the best uses of this phenomenon include CTscanning of the injection site for confirmation of optimal localizationor actual CT guided placement of the injection where necessary withimmediate confirmation of location and dose amount delivered. Thesuperior spatial linearity of CT compared to MR, makes CT preferable forstereotactic placement tests. CT is also effective for these agents atthe concentrations achieved in lymphatics after subcutaneous orintramuscular injection.

When an alternating magnetic field is applied to a magnet, a number ofresonant interactions can come into play which can completely destroythe net magnetization. The main resonance is to do with the precessionfrequency of the dipoles around the main axis. There are also resonanceeffects in bulk magnet to do with movements of the Bloch walls betweendomains as well as with the size of domains. In a sub-domain sizedsuperparamagnetic particle, the principal determinant of resonantbehaviour is generally the intrinsic flipping frequency due to thetemperature, particle size, and compositional anisotropy. Exploration ofthe impact of radiofrequency signals on the resonant behaviour ofsuperparamagnetic particles of well defined sizes is suggestive ofnumerous useful effects.

For more specific separation of the particles according to resonantbehaviour, a chromatography column or a very long coil of narrow boretubing can be placed inside a high field magnet such as a 2.0 Tesla MRImagnet, and the column then surrounded by an elongated solenoid coilwith various switchable capacitors, resistors, and inductors attached.This apparatus can be used to subject the chromatography column to aseries of selected radiofrequency fields. During the irradiation of thecolumn with a particulate field frequency, those particles that arerelatively demagnetized at that selected frequency will commence movingdown the column while the remainder of the assortment of particles willremain fixed in the external magnet's field. That fraction of resonantselected particles is collected, and then the frequency of the appliedfield is changed to permit elution of a second resonant selectedfraction, and so on in this fashion until a series of different resonantselected fractions are collected. The demagnetization can be achievedeither by pulsed RF irradiation which flips the coherent particle axisinto a transverse orientation, or, more efficiently, by introducingsufficient energy to induce non-coherent flipping of sub-unit dipoles.

By the use of these alterations in size, composition and intrinsicresonant magnetic behaviour, a series of particles is produced withdiffering resonant behaviour which can optimize them for use in an MRIdevice of a given field strength and proton Larmor frequency. Also, byproducing highly purified resonant engineered particles in this fashion,it becomes possible to produce the phenomenon of SelectiveRadiofrequency Flipping Alteration (SRFA). The resonant engineered,purified particles are subjected to a selected radiofrequency signal (bymeans of additional coils around the imaging subject) and sufficientenergy is introduced to overcome the coherent alignment of thecrystalline sub-units with the applied external field. This results inan effective demagnetization of the particles and a sudden reduction oftheir contrast effect in an MR image.

Two MR images may then be collected a few hundred milliseconds apart,with the first being contrasted and the second being non-contrasted.These two images are then subtracted from one another by the computerand a substraction image results. This yields a “contrast neurography”by which only the nerves and any other tissues with high concentrationof the particles are seen. The process can be repeated at a differentappropriate frequency for each type of resonant tuned particle injected.In this fashion, several different nerve roots could be visualized, eachin a different image if their respective muscles of innervation had beeninjected with different resonant tracers. The lymphatics will collectall the tracers and so will be subtracted from all the images.

This SRFA subtraction technique may also be applied to other ferrite MRIcontrast methods such as antibody based labelling of tumours orinfection sites wherein several different antibodies could each beattached to a different resonant particle group. Then by using anapparatus that can generate multiply tuned frequencies within the MRmagnet during imaging to serially change the frequency at which thesubtraction image is obtained, the external images can be used todetermine which antibody is adhering to the area of interest.

Another distinct use of these resonant modifications is to prepareparticles with frequencies in the microwave range. Such particlesexperience mechanical vibration and hence hearing when subjected toresonant tuned microwave energy in this method, the particles would betransported into areas of spinal cord injury where the development ofscar prevents the regeneration of injured spinal cord tissue. Inresearch work, this localized heating phenomenon might be used as ameans of inhibiting spinal cord scar formation. This effect may also beapplied for selective tumour diathermy. Intramuscular injection atvarious sites with different resonant particle frequency types at eachsite will permit rotation of microwave heating frequencies so that onlythe tumour site will be stimulated by all the signals.

From the above, it will be appreciated that the method of the inventionprovides an entirely novel means of pharmaceutical distribution whichinvolves the entrainment of a well known physiological phenomenon calledaxonal transport. A central feature is that the total body distributionafter intramuscular injection of the pharmaceutical agent quiteunexpectedly yields dramatically high intraneural concentration relativeto other tissues. This differential in body/nerve concentration permitsthe use of this route with relatively small amounts of pharmaceuticalagent to achieve nerve based imaging and treatment effects.

Insofar as the method of the invention is concerned, it may be helpfulto review the background to the present understanding of axonaltransport processes.

A neuron which innervates a muscle in the human foot is an enormoussingle cell (see FIG. 5) nearly three feet in length whose nucleus inthe spinal cord must manage chemical metabolic events taking place faraway in the axon terminus. The supply of newly synthesized proteins,membrane vesicles, and organelles such as mitochondria is accomplishedby first producing these items in the cell body, then transferring themalong the axon at rates of up to a meter per day. This ‘anterograde’flow could result in a tremendous accumulation of material in the axonterminus unless compensated by a return or ‘retrograde’ flow at similarrates and by a similar mechanism.

Although there are various rates and mechanisms of axonal transport, thefast anterograde and retrograde flows (see FIG. 11) are carried out bymotile proteins (kinesin and dynein respectively) which drag moleculesand vesicles along the microtubules of the axoskeleton. The materialstransported include not only structural and metabolic molecules, butalso molecules sampled from the external environment of the axonterminus which are passed back up to the neuron cell body to inform itof the environment. Such signals include various trophic or growthfactors originating in cells near the axon terminus which areendocytosed by the axon, encapsulated in lipid vesicles, and varioustrophic or growth factors originating in cells near the axon terminuswhich are endocytosed by the axon, encapsulated in lipid vesicles, andthen passed up to the cell body for processing or analysis via theaxonal transport system (see FIG. 12).

The rate of transport of a given substance is independent of electricalactivity within a neuron but does vary with the type of molecule beingtransported. Anterograde axonal transport has a major fast and a slowcomponent. The slow component is divided into “slow component a” and“slow component b” at rates of approximately 1 and 3 mm/dayrespectively. These slow components apparently reflect gradualstructural repair and replacement of the subunits of the cytoskeletonand are not involved in the fast components important for tracerstudies.

The fast component of transport demonstrates distinct maximal rates foranterograde (300-400 mm/day) and retrograde (150-300 mm/day) transportand some rates up to a meter/day have been reported. The maximal ratesof transport apply to small membrane vesicles. Further, there are avariety of “waves” or distinct sets of slower transport rates exhibitedin characteristic fashion by various molecules.

All of this movement is ATP and calcium dependent. The metabolisminvolved is local, i.e. mitochondria bound to the axolemma as well asmitochondria being transported on the microtubules use glucose andoxygen absorbed through the cell membrane along the axon to generate ATPlocally.

The existence of axonal transport (or ‘axoplasmic flow’) has been knownfor over 40 years and it has been known for twenty years that certainforeign materials injected into muscle would be endocytosed (swallowedup) by the axon terminus and then subsequently be detectable in theneuron cell body; however, until the developments described herein, allmethods of detection have required lethal interventions, generallyrequiring the killing of the experimental animal with subsequentspecialized tissue processing.

A series of relatively non-specific substances for uptake were triedincluding Evans-Blue stain conjugated to albumin and also horseradishperoxidase (HRP) enzyme, and radio-labelled amino acids for anterogradelabelling. The principal of improving specificity and uptake efficiencyof a histologically identifiable tracer was taken further by Schwab(Brain Res. 130:190-196 (1977)) who attached nerve growth factor (NGF)to HRP. It was also Schwab who showed that a plant lectin called wheatgerm agglutinin (WGA) was an excellent nerve adhesion molecule and againSchwab who introduced the use of viral fragments and toxins as labels(see Brain Res. 152:145-150 (1978) and J. Cell Biol. 82:798-810 (1979)).

WGA conjugated to HRP was later suggested as a tracer and this one agenthas been the predominant agent of choice in many hundreds of subsequentstudies involving axonal transport. The conjugation to somepost-sacrifice visualization moiety such as HRP permitted the use of achromogen histochemical staining reaction. Other means of visualizationof tracers included autoradiographic histology or immunocytochemicaltechniques.

Once endocytosed, WGA-HRP conjugates are found in Golgi/EndoplasmicReticulum/and Lysosomes (GERL) and are transported at a slower rate thanHRP alone. Many of the agents which employ plant lectins, viral toxinsand surface fragments, and some anti-synaptosomal antibodies astargeting moieties are taken into the cell by “adsorptive endocytosis”.

There is also a route called “transcytosis” taken by unconjugatedlectins. These molecules also bind to receptors before endocytosis butare then transported within the cell without being first introduced intolysosomes. This mechanism has also been shown with a monoclonal antibody(“192-IgG”) raised against an NGF receptor on pheochromocytoma cells andhas made it possible to show that the NGF molecule binds to the receptorprotein and that the entire complex is then transported up the axon tothe cell body.

Another interesting ligand/receptor complex involves [H³]-Lofentanil andthe opiate receptor which are endocytosed and transported by sensoryneurons. PET studies with [C¹¹]-carfentanil have been used to assess thegeneral distribution of opiate receptors, but this approach has neverbeen tried as a means of tracing selected tracts via axonal transport inhumans. Similar studies with GABA, D-aspartate, dopamine,norepinephrine, and serotonin have shown that uptake and transport ofneurotransmitters is a widespread phenomenon in the CNS as is thetransport of receptors.

Acetylcholinesterase uptake and transport has been studied for manyyears because of its ease of use as a histochemical marker. Otherstudies have demonstrated transport of a wide variety of substancesincluding Vasoactive Intestinal Polypeptide (VIP), cholecystokinin,substance, P and somatostatin, neuropeptide-Y, and adriamycin. Thesetypes of tracers have sometimes been introduced by intravenous injectionwith subsequent uptake by neurons as well as by actual tissue injectionin or near the neurons of interest.

Yet another set of studies has involved neurotrophic viruses such asHerpes Simplex, poliovirus and bacterial neurotoxins, e.g. tetanustoxin. Of the various tracers, tetanus toxin is the most effective for“transsynaptic” labelling in which the next neuron in a synapsing seriesis also labelled. It is possible that killed vaccines, or toxoidversions of these could be useful. As with physiologic molecules, theyoffer high avidity for the neuron and their transport kinetics have beenpreviously studied.

Another important phenomenon is transneutonal transport wherein traceris apparently extruded back onto the cell surface after transport thusacting to produce a sort of second injection at the next synapse in thechain (see Gerfen in Exp. Brain Res. 48:443-448 (1982)). Tetanus toxinappears to move in a specifically transsynaptic fashion, but WGA andWGA-HRP are found in glia after anterograde transport of WGA-HRP, andsynaptic structures need not therefore be involved.

Another area of advance has been in the use of particulate tracers.Olsson in Neurosci Lett., 8:265 (1978) suggested the use of anon-specific very small particulate iron-dextran complex in which theiron was in gamma iron oxide form and in which post-sacrificialdetection involved microscopic study after chemical staining for iron.Other important particulate tracers used for histological light andelectron microscopy have included a large protein with a ferritin core,1-10 nm non bio-degradable colloidal gold particles and colloidalfluorescent particles some 15-20 nanometers in diameter. Latexmicrospheres with fluorescent labels and ranging from 50 to 200nanometers in size have also been used. However, there has been acontinuing belief that larger particles can only be transported afterneuronal injury and most of the particle studies have involved transportbetween locations in the central nervous system after traumatic needleinjection into the brain substance (see Colin, Brain Res. 486:334-339(1989))

Detection of transport in living neurons has been accomplished inseveral ways. Thus for example, the neuron may be rapidly removed intactfrom the killed animal and placed over a series of proportionalβ-particle counters to detect the passage of a radioactive tracer pulsealong the axon. It is also possible to directly observe the movement oforganelles along such excised neurons via microscopic video interferencecontrast techniques.

There is however no prior art for in vivo imaging use of nerve adhesionmolecules coupled to clinically imageable a tracer molecules which doesnot involve direct inspection of neural tissue. Further there is noprior art for any entrainment of axonal transport to achieve desireddistributions of any actual pharmaceuticals for human or veterinarytherapeutic use. Axonal transport has been much studied as aphysiological process (analogous to the study of DNA prior to the adventof industrial biotechnology) and it has been used extensively forstudies in which the delivered agent is effective only after the deathof the organism (as in histology) or achieves its effectiveness onlythrough the killing of nerve cells which transport various toxins.However, there are no prior clinical uses, or uses in which the effectis achieved in a living animal or human with intended diagnostic ortherapeutic rather than neurotoxic effects.

Very recently (after the priority date hereof), Brady SMRM 10:2 (1991)verbally reported transport of MR detectable particles after directinjection into the sciatic nerve; however he exhibited only an image oftransport after the completely severed sciatic nerve was soaked in a gelwith ferrite particles. Ghosh in SMRM 10:1042 (August 1991) similarlyreported evidence of transport of ferrite particles after directpressure injection into the brain of a frog, although no MR detectionwas achieved. Neither taught how pharmaceutical use of axonal transportcould be achieved since these techniques involved irreparabledestruction of vital neural tissue. Intraneural injection is destructiveof the nerve at the site of needle puncture and causes forced flow oftracer in the nerve sheath which may actually mask evidence of actualaxonal transport. Madison in Brain Res. 522:90-98 (1990) also reportedpressure injection of latex nanospheres into the brain wherein thespheres were used to deliver toxic agents for the killing of neuronsafter subsequent photoactivation. These reports can, indeed, be taken asevidence of the non-obviousness of the non-destructive techniquesdescribed herein.

Non-destructive administration of toxic anthracycline antibiotics hasbeen reported, but this was done to study the chemical nature of theneural uptake process and the fluorescent effect of the agents ratherthan to achieve any therapeutic effect, and the agents concerned wereneurotoxic (see England in Brain 111:915-926 (1988) and Bigotte inNeurology 37:985-992 (1987)).

Unlike any of these previous reports, the agents described herein may beintroduced by techniques which do not involve the destruction of neuraltissue and which then achieve a pharmacologic effect which does notrequire any toxic injury to neural tissues. By delivering particulatecarriers it becomes possible to deliver types of pharmaceutical agentswhich would be irreparably damaged by direct chemical conjugation to aNAM or on break up of its direct NAM-conjugate within the cell. Insteadthe NAM is coupled to the particle and the drug is included in theparticle or in the particle coating. Further, the use of particulatedrug carriers permits the introduction of large numbers of molecules ofthe pharmaceutical agent with each endocytotic event thus yielding a 100fold or up to one million fold increase of uptake efficiency per NAM.This amplification effect may be crucial to achieving pharmacologicallyefficacious doses in many situations. The methods of administration forthese beneficial diagnostic and therapeutic uses include topical,intravenous, intrathecal/intracisternal (cerebro-spinal fluid),sub-cutaneous, intradermal, intra-nasal, eye-drop, or bladder irrigationmethods, but intramuscular administration is to be preferred.

The agents described herein differ from all previously used axonaltracers in that they include agents capable of controlled administrationby safe intramuscular injection with non-toxic substances and ofachieving whole body distributions which permit their useful observationby various types of non-invasive imaging modalities. The agents may bebiodegradable, safe for clinical use, and act to reveal various humandisease conditions which cannot be adequately demonstrated by existingtechniques.

Previous uses of axonal tracers have been concerned with optimizing thedegree of post-sacrificial staining of the neuron cell body in brain orspinal cord. It has not previously been evident that usefulconcentrations and distributions of clinically applicable tracermaterials could be achieved.

However, this set of agents is based on the discovery that when a nerveadhesion molecule which also has affinity for markers on the muscle cellsurface is used, the injected material has very minimal spread from thesite of intramuscular injection. In consequence, a relatively largeamount of the substance is transported into the nerve while relativelylittle spreads throughout the body. The initial distribution assayresults with animal studies using ¹²⁵I labelled WGA are shown in FIG.13. This showed that the concentration in peripheral nerve was up to tentimes higher than in any other tissue excluding the site of injection.The injection site could be masked out of an image so this suggestedthat the distribution after intramuscular injection might be consistentwith imaging.

However, although the concentration in the nerve was 10 to 50 timeshigher than for example in surrounding muscle, the total volume of thenerve relative to the volume of surrounding tissue was quite small.Thus, only an imaging technique which could collect signals from a verysmall ‘voxel’ size could successfully recover the signal. At thisrelative concentration, simple labelling of a small molecule or proteinwith a gamma emitter for SPECT detection would be inadequate.Substitution of a relatively long half life positron emitter (¹²⁴Iodine)for the ¹²⁵Iodine would provide nearly adequate voxel size but wouldinvolve substantial spread of radioactive iodine through the body. Otherrelatively long half life positron emitters presented similar problems.

The use of a magnetic resonance small molecule contrast agent such asgadolinium-DTPA (diethylene-triaminepentaacetic acid) required theintroduction of a very high concentration into the nerve and this amountwas beyond what could be achieved. However, by synthesizing aparticulate magnetic resonance contrast agent based on a ferrous ferritecore, coated with dextran and conjugated to WGA, a series of useful,solutions to the problem were revealed.

Very surprisingly, the distribution results with even a crudepreparation of this type of agent which was not affinity purified wereup to an order of magnitude better then the previous results withiodinated WGA (I¹²⁵-WGA). The concentrations in nerve were 50 to 100times higher than in any other tissue excluding the injection site andlocal lymph nodes (see FIG. 14). However, unlike the I¹²⁵-WGA result,the concentration in the nerve was actually considerably higher than inthe neuronal cell bodies of the spinal cord. This distribution willoften be advantageous since most of the metabolism of the particlecarrier will take place in the nerve and surrounding Schwann cells whilepassing mostly only smaller molecules on to the cell body in the centralnervous system. Using highly purified, affinity specific product,exceedingly desirable distributions result, with effectively nildetectable agent in any tissue, but for traces in liver despite verygood intraneural concentrations. Non-specific particles eluted from theaffinity column without using the affinity eluant, but injected inidentical concentration and amount yielded no evidence of axonaltransport only particulate tracer conjugated to affinity purified NAMentered the nerve in high concentration.

Using ferrite doped polyacrylamide gel phantoms, it was observed thatthis preparation could reduce the T₂ relaxation time of nerve below 30milliseconds if the intraneural concentration of iron were greater than5 micrograms/ml. The injections with the crude preparations actuallyachieved concentrations in nerve of over 50 micrograms/ml (see FIG. 15).An experimental imaging magnet was modified to carry out confirmatorytests which permitted an image resolution with voxel size of only{fraction (1/10)} of a millimeter and using this system it was possibleto measure and callibrate nerve contrast distinguishing the tibial nerveof the injected from the uninjected leg (see FIGS. 16 to 20).

Nerves which were subsequently excised and measured for exact T₂ in themagnet confirmed the desired 50% reduction of T₂. Electron microscopyconfirmed uptake and transport of the intact particles (see FIGS. 21 and22) and the T₂ results showed that their rate of metabolism in the nervewas slow enough for their superparamagnetic properties to be maintaineduntil the time of imaging. The electron microscopy also revealed thatmost of the particles were being passed out of the neuron into theendoneurial fluid surrounding the nerve. This export of the tracer wasaccomplished by the paranodal complex at the nodes of Ranvier (see FIG.10). From the endoneurial fluid, the particles were being attached tothe outer surfaces of the Schwann cells which surround the axon due toaffinity of the WGA label for the Schwann cell surface.

In parallel with these studies, a positron emitter, ⁵²Manganese, wasused to make spinel moderated positron emitters and these were preparedin gels to duplicate the concentrations achieved with ferrous ferrites.This study confirmed the physical prediction that with as low as 25:1contrast ratio, a 1 mm object could be readily detected anddistinguished from a larger object simulating a lymph node onecentimeter away (see FIG. 23). Thus, the SMPE version of the agent wasshown to be adequate for PET observation of the transported agent. Thesedistributions also permit the use of SPECT labelled crystals for nerveimaging studies in humans.

The delivery of particulate pharmaceuticals by the intraneural route isan entirely novel means of drug administration. The largest number ofdrugs in current use depend in some way upon the bloodstream to achievetheir distribution. This vascular dependence includes not only drugsgiven by intravenous or intra-arterial routes, but also most orallyadministered drugs which must be absorbed into the bloodstream to reachtarget tissues, most intramuscularly administered drugs which areabsorbed by the muscles blood vessels, many inhaled agents, mostintranasally applied drugs, some rectally administered drugs such asparaldehyde, and many topically administered agents such as transdermalnitroglycerine. There are, however some drugs which are delivered intoand distributed by the cerebrospinal fluid (intrathecal route), someoral drugs which are not absorbed (kaolin, oral vancomycin), a varietyof topical and intravaginal agents, and some administered percutaneouslyfor local effect or intraarticular effect such as local anaesthetics,and locally administered steroids,

Various new drug forms and methods of use described below involvedelivery via an intraneural route. Access to this route may be obtainedby oral ingestion, topical, intra-articular, intrathecal, intravenousand, preferably, intramuscular administration. However, common to allthese new methods, is that the dosing and active site of the agent isdetermined by a route which involves endocytosis by nerve endings withsubsequent transport to a different and distant part of the neuron.

In some experimental studies, various agents for transport have beenintroduced by intraneural injection or by application to the cut end ofa severed nerve. In these methods, the blood brain barrier due to theperineurium is traversed by mechanical injury, and much of the uptake oftracer is due to direct presentation at cut nerve endings where specificnerve adhesion molecules may be irrelevant. The intramuscular technique(and also the intravenous application) depend upon the natural defect inthe perineurium which occurs at axon terminus. Thus the blood brainbarrier is naturally incomplete at this site so that tracers reversiblyadherent to muscle or emerging from small blood vessels passing nearneuromuscular synapses can present a variety of molecules and particlesdirectly to the neuronal cell surface for uptake after specific adhesionto the neuronal cell surface at the axon or dendrite terminus.

The site of injection or administration will preferably be determinedonly by knowledge of the nerves which project to that site. For example,a pain in the large toe will be known to the neurologist to involve thedorsal root ganglion of the fifth lumbar nerve root. He will then choosean injection site somewhere in the dermatome or myotome served by thatnerve root in order to label the part of the nerve he believes to beimpaired or to deliver, for instance, a pain medication to the ganglionor spinal cord dorsal root entry zone connected to the fifth lumbarspinal nerve root. For radionuclide imaging as well as for drugs wheresytemic spread is to be minimised, it is particularly important to beable to achieve high uptake by neurons per unit amount injected and tominimise spread away from the injection site.

When imaging is done, the image will preferably be collected at highresolution of a site which is different from the site of injection, butwhich is connected to the injection site by a nerve. The imaging willalso be done at a time which allows the agent to be transported thenecessary distance from the injection site to the active or imaging siteat a natural rate related to the size and type of the injectedintraneural drug.

Where the intraneurally administered agent is a negative (T₂-reducing)MRI contrast agent, contrast may be further enhanced by administrationof a positive MRI contrast agent (e.g. Salutar's SO41, Squibb'sPro-Hance or of course Schering's Magnevist) so as to distribute intothe tissues surrounding the neuronal pathway under investigation. It hasbeen shown that STIR and CSI (Chemical Shift Imaging) sequences helpsharply distinguish nerves from any surrounding fat and so emphasise theimpact of the contrast agent. For example a STIR (Short Tau InversionRecorvery) with tau=160 ms, t_(c)/2=30 ms and a long d₁=2 seconds todecrease saturation will accomplish this desirable effect to bestdemonstrate&the axonal imaging effect of ferrite tracers.

The nerve adhesion molecules used to initiate the uptake and transportof the pharmaceutical agents used according to the invention all have incommon with each other some tendency to promote uptake by neurons. Somemolecules with no particular affinity may be taken up and transportedinefficiently by neurons. However, molecules which interact with andbind to specific cell surface markers or receptors on the nerve endingof the selected nerve type are far more efficient and are preferred. Anadditional degree of efficiency can be obtained when the compound alsohas some affinity for the cell surface of muscle cells, since this willpromote the depot effect at the injection site and decrease the tendencyfor the agent to diffuse away or be carried away by the bloodstreamprior to uptake by the neuron. However, in some applications, such asSPECT and PET imaging, it may, in fact, be desirable to encourage suchwashout by the bloodstream to minimise radiation dose to the muscleafter a brief period of uptake by the nerves innervating that muscle.

For investigative work involving animals, wheat germ agglutinin (WGA)can be used to provide specificity for active uptake and transport.However, there are a wide range of nerve adhesion molecules which can beused to cause selective and active adsorptive endocytosis into nerves.This class of nerve adhesion molecules includes:

1) Anti-synaptosomal monoclonal (and non-monoclonal) antibodies whichare purified or generated based upon their affinity for nerve membranes.These can be made by using crude nerve homogenates and then testing forendocytotic efficacy in cultured neuroblastorna cells or by directmeasurement of uptake of radiolabeled forms after intramuscularinjection in laboratory animals. These agents may involve entireantibodies of, preferably, the fragment of the antibody responsible forrecognition without the F_(c) region. Similar considerations apply forantibodies to dopamine-beta-hydroxylase.

2) Various growth factors such as nerve growth factors, epidermal growthfactors, insulin-related growth factors and other proteins and peptidesin this functional category which are known to have or discovered tohave efficacy at causing the neuronal uptake of themselves and of otheragents with which they are conjugated.

3) Lectins of various sorts which are proteins having a high degree ofaffinity for particular carbohydrate and other types of cell surfacemarkers. This is meant to include both plant lectins as well as variousendogenous vertebrate, mammal, or human lectins as are known or may bediscovered.

4) Fragments of neurotrophic viruses such as Herpes simplex,pseudorabies virus or poliovirus or proteins or other markers from theviral coat responsible for their highly efficacious uptake andtransport. These fragments or inactivated whole viral particles, orcloned and produced copies of the crucial proteins are of particularinterest for trans-neuronal transport.

5) Fragments of bacterial toxins such as the B-chain of cholera toxinand non-toxic fragments of tetanus toxin such as the c fragment as wellas modified versions or cloned portions of other safely administeredproteins with high neural affinity.

6) A wide variety of peptides and small proteins such as endorphins,vasoactive intestinal polypeptide, calcitonin gene-related peptide,cholecystokinin, substance P, somatostatin, and neuropeptide Y or therelevant portions of such peptides for the encouragement of neuronaluptake and transport.

7) Enzymes which are selectively endocytosed for synaptic recyclingpurposes such as acetylcholinesterase and dopamine beta hydroxylase orportions of these enzymes which are effective at inducing neuronaluptake.

8) Various cell adhesion molecules including peptides, proteins andvarious simple and complex carbohydrates which are effective atpromoting neuronal uptake and transport of conjugated pharmaceuticalagents.

9) Neurotransmitters and neurotransmitter analogs such as GABA,D-aspartate, dopamine, norepinephrine, serotonin, and benzodiazepinedrugs which can be so constructed as to maintain their efficacy inpromoting transport after the conjugation to pharmaceutically usefulagents.

Two optimal nerve adhesion molecules for most applications aretransferrin and β-nerve growth factor depending on whether primarilymixed or primarily sensory nerves are to be imaged; however, variousother molecules may be optimal for particular sorts of pharmaceuticaltasks.

These proteins and other nerve adhesion molecules can be attached to thetherapeutic, prophylactic or diagnostic moiety or to the coating (e.g.dextran coating) thereof by various methods including a periodateoxidation reaction carried out under mild conditions which does notaffect the oxidation state of ferrite crystals or the binding affinityof the targeting molecule. Other binding reactions include carbodiimidebinding, glutaraldehyde binding, biotin/avidin linking, or noncovalentcoating with the molecule of interest. Metal oxide particles provide acore of useful size which can be securely coated by a variety of typesof agents under mild, non-denaturing conditions—they are thereforeuseful as a particle seed even when their special metallic or magneticproperties are not relevant to the task at hand.

All of the above conditions may be optimized so as to use this new drugadministration method to efficiently deliver a specially designedpharmaceutical to a specific and preselected location within the nervoussystem. The sites will include peripheral nerves along their continuouslengths, sensory ganglia, autonomic ganglia, spinal cord, and brainstemor the olfactory tract system upon intranasal administration. When theroute of administration is intrathecal, or topically upon the brainduring surgery or in the brain during open surgery or duringstereotactic surgical procedures, then a wide range of sites within thecentral nervous system can be reached. In these cases of directapplication in the brain, it may be the purpose of the agent to betransported to a surgically inaccessible site by axonal transport from asurgically accessible site, or in any case to a site different from thesite of application. This may also be used to demonstrate a successfulincorporation of grafted neural tissue which will become capable oftransporting included agents from the graft to distant sites. It willoccasionally be helpful to achieve transport with a small molecule suchas an amino acid or protein labeled with e.g. a positron emittingnuclide; however, in many of the applications, the preferred type ofagent will include a particle which is up to 50 nanometers in diameterand may contain many atoms, crystal subunits, or molecules of the activeingredient for diagnosis or therapy.

The exact relation between particle core size and resulting T₂relaxivity per microgram or iron is dependent on a variety of factors inthe particle synthesis and upon the biochemical environment in which theparticle must exist prior to imaging. Shen in SMRM 10:871 (1991) reportsa relation between relaxivity and crystal size which greatly favours theuse of the largest possible particle. Relaxivity data on particlesproduced by the inventor showed values of up to 4.0×10⁵ sec ⁻¹M⁻¹ whichare greater than those reported by Shen and are consistent with thistrend. Thus, an optimal relaxation effect per amount of iron to becleared by the nerve may be achieved with the largest particlesendocytosed and transported efficiently.

When more diffuse effects are desired, an injection into the bloodstreamor into the cerebro-spinal fluid can be used to cause uptake by aspecific type of neuron wherever it contacts the fluid. However, forspecific delivery to an individual peripheral nerve, the intramusculartechnique will be preferred.

The usefulness of the intramuscular administration derives from the factthat individual axons which enter a muscle divide multiply and fan outto innervate numerous individual muscle cells making up a ‘motor unit’.Such a motor unit is a group of muscle cells which fire simultaneouslywhenever their shared axon delivers a depolarization signal. Due to thedictates of muscle energetics and control mechanisms, the cells of aunit are generally scattered through a muscle rather than concentratedin a single area. Thus an injection at any one site will usually be nearthe axon termini of a large number of different motor units (see FIG.7). This presents the injectate with a relatively large axon terminalsurface area and provides access to a mix of axons distributed evenlythrough the incoming nerve. Additionally, because all muscles have arich sensory supply to muscle spindle tension, length, and ratereceptors, distributed through the muscle, there is simultaneous accessto sensory and to motor fibers (see FIG. 8).

When I¹²⁵-NGF was injected in muscle, high intraneural concentrationswere achieved because of uptake by the various types of sensory endingsin the spindle organs of the muscle.

Intramuscular injections with intraneuronal pharmaceuticals present nogreater risk of nerve injury than any other sort of routineintramuscular injection. However, it is well documented that any fineaxonal branch which is injured causes no lasting muscle impairment. Thisis because of the short distance of regrowth and the tendency for axonterminal branches to recolonize any denervated muscle cell. It should benoted that although injury to some axonal branches may occur, thedistribution of tracer in nerve and in cell bodies upon histologicinspection of the spinal cord is consistent with uptake by intact cellsrather than by any important contribution from uptake by injured cells.

There are a wide variety of clinical uses for an imaging method thatlabels nerves, traces connections and assesses pathologic changes inneurons. Axonal transport continues in crushed or compressed nerves withaccumulation of transported substances both proximal and distal to thesite of injury and so could be used to mark the location of thepathology—inside or outside the spinal cord. Experiments conducted bythe inventor confirm the accumulation of ¹²⁵I-labelled WGA distal tocompression and ligation sites at concentrations just moderately greaterthan in distal nerve but much greater than in proximal nerve, withrelative concentration ratios compatible with image based location ofinjury sites.

Imaging of axon transport may be achieved using agents in which variousnerve adhesion molecules including proteins capable of inducing activeendocytosis by axon termini are bound to coated mixed metal spinelparticles or other particles or diagnostic markers optimised for thevarious techniques discussed herein or to such small molecules ordetectable nuclides as are suitable to the imaging method of choice.After administration by an appropriate route, such as intramuscularinjection, the compounds are caused to enter and to travel along withinthe nerve axons. The alteration in imaging contrast progresses along thenerve from the axon terminus towards the cell body (or vice versa)unless it is impeded by a nerve compression or crush. In an incompleteinjury, the tracer molecule will accumulate at the blockage point. Therate of accumulation will be affected by such phenomena as turnaroundtransport at the compression site and by depression of rates oftransport in more severe compressions. In general, the administration ofthe agent is intended to produce a change in relative concentration ofthe agent which will distinguish axon contrast proximal and distal tothe site.

The rate of progression of the contrast particles is determined in partby their size and by the particular protein used to provide theirspecificity. Attachment to nerve growth factor as opposed to other celladhesion molecules will take advantage of specialized transportpathways. Various specific neural pathways can be studied by using e.g.antibodies to the opiate receptor or to other receptor/neurotransmittersystems or even to non-receptor synaptosomal antigens as the proteinportion. However, for a given adhesion protein, the rate of progressionof the particles along the axons will be altered in various neuropathiesand other diseases affecting nerves.

Intramuscular injection of 10 to 100 microliters of concentratedparticles (5-20 mg Fe/ml is achieved by use of Amicon Centriprep-30concentrators or by reconstitution after freeze drying) is adequate tocause transport which can then be imaged. Much smaller injections can bemade into central nervous system tissues under stereotactic or imagebased guidance in order to observe transport between central structuresor even for drug delivery. Also, using proteins which encouragetransneuronal transport, a peripheral muscle injection can be used tocause transport along the spinal cord and so help to diagnose theseverity of spinal cord injuries.

The various types of inorganic particles described herein can all beattached to nerve adhesion molecules for the study and evaluation of thenervous system by means of axon transport. These materials include 10 to50 nm ferrous ferrite dextran coated particles, resonant tunedsuperparamagnetic particles, enhancement agents for Overhauser MRI (seefor example WO-A-91/12024), multiple nuclide particles for MRspectroscopic and multiple nuclide MR imaging, positron emittingparticles, gamma emitting particles for SPECT, proteins or smallmolecules labelled with positron or gamma emitting nuclides, shieldedpositron emitting particles, high Z substituted particles for CT X-raycontrast with poly-energetic or selective mono-energetic imaging.

Additional types of agents for imaging include paramagnetic metalchelates of polychelants (e.g. poly-lysine gadolinium-DTPA 40 which usesthe macromolecular/particulate aspects of uptake to introduce groups ofparamagnetic nuclei (40 Gd atoms per molecule) (see EP-A-305320,EP-A-357622, EP-A-355097, EP-A-331616, WO-A-90/12050 andWO-A-90/13256)), liposomes containing superparamagnetic or paramagneticMR contrast compounds, and air containing albumin spheres typically usedfor ultrasound contrast which can introduce susceptibility based MRcontrast effects into nerve with a minimum of foreign material todigest. Also, fluorescein or other biocompatible fluorescent moleculesconjugated to a nerve adhesion molecule or conjugated to dextran coatedferrites can be injected to permit confirmation of nerve location by aneurosurgeon during an operation. Spinal Root Compression from HerniatedLumbar Disk: The axon transport of ferrites or other particulate agentsaccording to the invention can be used instead of myelographic X-rayunenhanced X-ray CT (computed tomography), electromyography (EMG), nerveconduction velocity (NCV) studies and somatosensory evoked potential(SSEP) to evaluate back and leg pain to check for sciatica. The patientreceives a very small intramuscular injection of the agent at theirdoctor's office one to three days prior to the imaging session. Theagent then travels up the nerve, and a moderate contrast change developsin the nerve along the path of transport. However, if there is anycompression of the nerve, the contrast agent piles up “upstream” of theobstruction. An imaging study is then obtained.

The resulting scan would show the precise location of the nerve rootcompression, and, by the amount of contrast agent piled up at the nervecompression site compared to the amount that passes, the severity of thecompression could be assessed.

Unlike myelography, there is no lumbar puncture, no need forhospitalization, and if the MR version of the imaging agent is used, noneed for any X-ray exposure. A single study shows the surroundinganatomy, confirms the actual nerve compression rather than relatednearby problems that might or might not cause actual compression, anddemonstrates the physiologic effect of the compression throughdemonstration of interference with axonal transport. This isparticularly helpful in MR imaging since the nerve is often compressedagainst bone and the bone itself does not show well on MR. The use ofMRI in the diagnosis of sciatica has been greatly hindered heretoforebecause this imaging technique reveals herniated disks in up to 60% ofnormal asymptomatic individuals. What is needed is a means of showingboth that there is a herniated disk and that it is actually causing anerve compression since surgical decision making requires knowledge ofboth of these findings. There is no special risk of failing to diagnosefar lateral disc herniations, and there is no need for uncomfortable andunreliable EMG, NCV, or SSEP studies.

Cervical Radiculopathy: Nearly identical arguments apply to thecondition call “cervical radiculopathyl” in which an intervertebral diskor bony spur in the neck pinches a spinal nerve causing hand, arm,shoulder and neck pain. Myelography in this condition is even moredangerous since it involves placing a needle in the high cervical spinalcanal. A nerve injury from a needle in the lumbar region will onlyexacerbate sciatica, but a spinal cord injury from a cervical puncturecan cause death or quadruplegia.

By making myelography unnecessary in the assessment of sciatica andcervical radiculopathy there would result a very large overall reductionin procedure costs and radiologist's time, and a saving of tens ofthousands of hospital admission days.

Nerve Entrapment Syndromes: There are a wide variety of nervecompression syndromes of which the most well known is carpal tunnelsyndrome. In that particular condition, a gradual thickening ofligaments in the wrist causes pain, muscle wasting, numbness andweakness in the hands affecting hundreds of thousands of patients eachyear. There are some eight or ten other similar conditions affectingvarious nerves in various locations about the body (thoracic outlet,supracondylar/Struthers ligament, anterior interosseous and posteriorinterosseous/arcade of Frohnse, cubital tunnel/ulner palsy, ulnarcompression in the wrist/Guyon's canal, suprascapular, meralgiaparaesthetica/lateral femoral cutaneous, saphenous, peroneal, and tarsalnerve compression syndromes).

These conditions are exceedingly difficult to confirm. The only reliablemethod for carpal tunnel is EMG and many of the ether conditions must beinferred from the clinical examination of the patient with subsequent“blind” surgical exploration. In fact many depressed patients filling upthe waiting lists at pain clinics and consuming a variety ofnon-efficacious medications actually have easily correctable nervecompressions. These compressions cannot be treated, however, becausethey can not be reliably diagnosed or located.

The axonal tracer method is exceedingly well suited to the diagnosis ofall manner of nerve compression syndromes. These patients rarely havecomplete denervation, so axonal transport still functions distal to thecompressional point. This is a realm where the competing method is EMG(a three month waiting list for this painful test is common in the UK)or, in many cases where there is no existing method at all forconfirming a clinical suspicion without surgical exploration.

A related area of use concerns cranial nerve compressing responsible fortrigeminal neuralgia, Glossopharyngeal neuralgia Torticollis,hemi-facial spasm, Vertigo/Meniere's Disease, and even essentialhypertension due to Vagal compression.

Incontinence and Impotence: Another extremely common problem which isexceedingly difficult for the physician to evaluate is urinaryincontinence and bladder dysfunction. It is en important to determinewhether there is any failure of the nerves involved in distinction froma mechanical failure. This is currently an extremely difficult problem.However, a few carefully placed injections would permit imaging studiescapable of identifying a variety of treatable causes. A similar set ofproblems also arises occasionally in the evaluation of male impotence.

Localization of Nerve Bruises and Lacerations: The, muscles of the faceare operated by a single nerve which is unfortunately subject to severebruising or even laceration at several points during traumatic facialinjury. A clinician is often presented with a patient who, after a blowto the face, has a risk of irreparable corneal abrasion because hecannot close his eye, a distorted and grotesque fixed facial droop, andan ongoing drool from the corner of a mouth he cannot elevate. Theproblem is that there is no way to locate the exact site of injury alongthe complex course of the nerve as it travels among bones, muscles,glands, arteries, and other structures. If there is only a bruise to thenerve, then it will recover on its own over months with no interventionrequired, but if the nerve is actually lacerated, it must be reconnectedsurgically on an urgent basis to minimise the risk of retractionrequiring subsequent nerve grafting.

Unfortunately, until now there has been no way to learn how severe theinjury is. Because of the impact on the patient's life if the need torepair is not appreciated, one might advocate surgical exploration anddirect inspection in all cases. However, because the exact location ofinjury could not be ascertained, this would require unacceptableincisions at multiple locations on the face and throat with danger to avariety of uninjured structures along the course of the nerve.

This is a frustrating clinical problem, and there is no current methodto locate or assess such an injury. Because axonal transport continuesfor several days even after the nerve is actually cut, the agentsdescribed herein would dramatically alter the situation. An injection oftracer into facial muscle could be undertaken immediately in theemergency department and imaging would be possible within hours becauseof the small nerve transport distances involved.

Such an investigation could show that contrast agent still passed thecompression point so that only a bruise was responsible or, even if itcould not prove the severity, it would show the surgeon precisely whereto look via a tiny incision. Similar considerations apply for traumaticnerve injuries at various locations around the body as well as to theproblem of distinguishing between spinal nerve root avulsion andbrachial plexus injury.

Assessment of Spinal Cord Injury: In spinal cord injury, it is oftendifficult to distinguish deficits due to direct damage to the cord fromeffects of nearby root compressions. Studies of cord injury per se couldbe approached in several different ways. An injection into muscle wouldlabel the motorneuron cell body, and transneuronal transport would thenintroduce tracer into descending corticospinal neurons. By injecting in.transversospinalis back muscles, the initial transport distance could bereduced to no more than two centimeters. A second approach relies on themultisegmental distribution of motorneurons projecting to back muscles.An injection would cause labeling of cell bodies in intact cord with acutoff in areas where neuron cell bodies were crushed or injured. Athird method would be to rely on proprioceptive sensory neurons many ofwhich do not synapse at the level of entry into cord, but project up tothe medulla before reaching the first synapse. For this approach,injection of an intervertebral joint capsule might be effective.

Experiments done by the inventor have demonstrated transport ofradiolabelled tracer up the spinal cord after intramuscular injection.This has been most successful with small molecule imaging agents such astransneuronally transported WGA and potentially with tetanus toxinfragments, either of which can be labelled with Iodine¹²⁴.

Myelography is considered dangerous immediately after spinal cord injurysince the changes in spinal fluid pressure that result from the lumbarpuncture can make the spinal cord injury worse. Further, it often failsto reveal details of the site of injury since swelling will tend toexclude dye from the area of injury. MRI is sometimes used, but theresults are difficult to interpret since the extra tissue water due toswelling often overwhelms other imaging information.

A variety of other more rare spinal cord conditions might also be betterstudied by application of this technique. These include congenitalanomalies of the spinal cord which lead to tethering and stretching ofthe spinal nerves, and also a variety of inflammatory or neuriticconditions such as transverse myelitis which may affect axonal transportin the spinal cord.

Evaluation of Neuropathies: Another clinically important aspect ofaxonal transport concerns peripheral neuropathies such as occur indiabetes. These are conditions which involve dysfunction of nervemetabolism rather than actual mechanical impingement. Diabeticneuropathy afflicts hundreds of thousands of diabetic patients. A commonoutcome is loss of sensation resulting in sores and ulcers of the feetand legs (occasionally becoming so severe as to set the stage forgangrene or to require amputation), difficulty with balance and loss ofthe ability to walk. It is difficult to learn how to treat thiscondition because there is no way to accurately follow its course and noearly warning of its onset. A diagnostic agent according to the presentinvention could be injected into muscle and its rate of progressevaluated with MRI or other imaging techniques. In this fashion, adiagnosis could be made, and the severity assessed. Such a techniquewould be a tremendous boon to research in diabetes and might help setthe stage for progress towards some medical treatment in the future.

This problem as well as a variety of other clinical entities such asamyotrophic lateral sclerosis which causes profound, even lethal muscleweakness, Alzheimer's disease—which condition is exceedingly difficultto diagnose, and neurologic deficits after shearing type head injuriesare all thought to involve disorders of axonal transport or relatedaspects of axon cytoskeletal function. An imaging study that assessesquantity and rate of transport can permit diagnosis as well as followthe progress of remissions and would also be quite helpful in research.

Oncology: Neuropathy is a complicating concern in the management andtreatment of cancer. This is because neuropathies that are due to thecancer itself may be confused with neuropathies caused by chemotherapy.Tumours or metastases of tumours can cause neuropathy by directmechanical nerve compression, however a number of cancers seem to causeneuropathy by paraneoplastic phenomena which are not entirelyunderstood. A wide variety of chemotherapeutic agents also causeneuropathies and this sometimes is the key limiting factor on maximalpermissible dose.

The oncologist is therefore often faced with a dilemma when a patientdevelops pain, weakness and paresthesias during the course of treatment.If the neuropathy is due to tumour progression, then increased therapyis indicated. However, if the neuropathy is due to the treatment itself,then those drugs must be abandoned or replaced. An axon tracer imagingtechnique would be helpful in identifying nerve and spinal cordcompression, in studying the puzzling paraneoplastic effects, and in thedevelopment, dosing and monitoring of chemotherapeutic agents.

Epilepsy: Another interesting possibility is a link between axonaltransport and epilepsy. Kainic acid is used to induce a murine model ofepileptic kindling in the hippocampus. It is well known that thisinvolves increased excitability of the involved cells. It has beenobserved that kainic acid also serves to block retrograde transport ofhorseradish peroxidase. If there is any association between epilepticfoci and altered axonal transport, this could lead to a means of imagingepileptic foci as part of, for example an operation for microelectrodearray placement done several days prior to the definitive epilepsysurgery. Intra-operative application of the tracer at the firstoperation would provide useful information for the second procedure.

Verification of Denervation: It is sometimes desirable to denervate astructure. The most common such situation is surgical vagotomy to treatulcer disease. In these cases it is essential to achieve total vagaldenervation in order to assure there is no further gastric acidproduction. However, because of the complexity of the vagal innervationit is often difficult to be certain if an adequate result has beenobtained. This may require the continued use of various kinds of testingfor acidity and the continued use of medications at considerable expenseand difficulty for the patient. A misjudgment may lead to death byinternal bleeding or gastric perforation. Oral administrations of axonaltransport imaging agents will permit repeated assessments of vagalinnervation of the stomach and upper GI tract as these can be absorbedfrom the gastric wall if vagal innervation is intact.

Intraoperative Nerve Identification: There are a number of situations inwhich the neurosurgeon is faced with extreme difficulty indistinguishing nerves from pathological tissues of roughly similarcolour and texture such as tumours or fibrotic fat pads. This includessurgery for untethering of lipomyelomeningoceles and surgery forremoving acoustic neuromas where the facial nerve passes through thetumour. In this latter situation for instance, a fluorescein conjugatedor chromophore conjugated, dextran coated ferrite with a nerve adhesionmolecule conjugated as well may be used according to the invention. Aninjection into the facial musculature is done preoperatively and an MRimage is obtained to demonstrate the course of the facial nerve throughor around the tumour. An appropriate ultraviolet or other light sourcecan be directed towards various areas of the tumour mass to permitdirect visual confirmation of nerve location by the surgeonintracperatively.

Clinical Research Uses: Outside of purely clinical issues, there are avariety of compelling areas of neurobiology research where thediagnostic agents of the invention can be used, e.g. intraoperativeresearch on the neurophysiology and distribution of speech areas inawake humans. Although neuroanatomical tract tracing studies have beencarried out in monkeys to identify connections among areas thought to behomologous to human speech areas, human studies are needed. An axonaltracer with an MRI detectable label might permit tract tracing studiesin humans in conjunction with the intraoperative recordings.

Therapeutic Uses: It is often necessary to administer drugs whoseintended site of action is in the spinal cord or dorsal root ganglia(DRG). However, in the past there has been no easy way of safelyadministering such drugs near their site of action. While injections arecommonly used to achieve local drug effects in muscle or in joints, itis exceedingly hazardous to introduce a needle percutaneously andblindly into the vicinity of the spinal cord or sensory ganglia.Therefore, in order to achieve pharmacologically efficacious doses ofvarious drugs at these locations it has been necessary to give very highsystemic doses by intravenous and oral routes, or by intramuscularroutes wherein the actual delivery of the drug depended upon vascularuptake from the injection site in order to achieve the best availabledistribution. Alternatively, cumbersome procedures in which specialcatheters are threaded into place near the spinal cord have beenundertaken with attendant dangers of spinal cord injury. There has neverbeen any route except by whole body vascular distribution to deliver anydrugs to ganglia.

However, by taking advantage of the drug distributions achievable byaxonal transport where particulates are used, a dramatic change inpharmaceutical practice can be achieved. When the desired drug istrapped in a polymer, liposome, or protein nanosphere with an attachednerve adhesion molecule, it becomes possible to carry out anintramuscular injection of an extremely small amount at a location towhich the desired part of the nervous system is connected by aperipheral nerve. Most of the injectate will stay in the muscle at thesite of injection while the drug particles are ingested by nerve endingsand transported to the ganglionic or spinal cord sites toward whichtreatment is to be directed.

Liposomes with phosphatidylcholine and phosphatidylethanolamine (e.g. asdescribed by Grant in Mag. Res. Med. 11:236-243 (1989)), derivatized forattachment of a nerve adhesion molecule can be prepared in the necessarysize range for efficient uptake and neuronal transport. In this fashion,a wide variety of hydrophilic and hydrophobic drugs can be packaged fordelivery to their intended sites of action while minimizing systemicdose.

The particles yield a large amplification of the uptake process sincemany drug molecules are ingested by the cell with each event and helpsminimize spread of the drug away from the injection site.

While there are a number of well known methods for producing particulatedrug carriers, many of these are not suitable for intraneural drugdelivery. Many of the known techniques are not applicable because theyresult in the production of particles which are far too large to permitaccess to the axon terminus in general and synaptic cleft in particularas needed to promote neural uptake. For instance, many polylactic acidparticles can only be produced in sizes near 100 microns which is threeorders of magnitude too large. Liposomes similarly tend to be too largeunless they are produced as small unilamellar vesicles (SUV) whichrequire harsh physical and chemical conditions for synthesis.

Polycyanoacrylate/vinyl particulates (many of which are termed “latex”microspheres or nanoparticles) can be readily produced in theappropriate size range, but require the use of organic solvents andother treatments which are destructive to many biological molecules oftherapeutic interest. Albumin or other protein microspheres of usefulsize can be produced from sonicated emulsions, but these requiredenaturing by heating over 100° C. or chemical crosslinking to achievestability and this similarly limits the range of potentialpharmaceuticals.

Metal oxide particles are particularly convenient as drug carriers. Theyreadily form stable colloids of appropriate size and can be coated by awide variety of molecules. When the particles are prepared by adding astrong base to the metal chlorides in saturated dextran, a strong bondbetween the particle and the dextran is formed. Subsequently, someproteins can be covalently bound to the dextran molecules. However, thistype of synthesis precludes the use of any drugs which cannot toleratethe strong acidity of the metal salts or the rapid shift to a strongalkali typically used in the precipitation reaction.

The predominant solution to this problem has been to precipitate theparticles with no coating, and then apply the coating at a later stepunder more mild conditions. However, whether NaOH or NH₄OH is used, andwhether the metal salts are added to the alkali or vice versa, uncoatedparticles always aggregate and despite high power sonication of theresuspended pellets, it is difficult to produce a stable colloid.

Precipitation by addition to ammonia is more effective than using NaOHbecause of a clear peptizing effect reconfirmed in experiments conductedby the inventor. Such uncoated ammonia precipitates become stable forcentrifugal ultrafiltration even without sonification. This preparationis sufficiently reactive as to permit reliably stable but non-covalentbinding of a wide range of molecules. In one series of preparations madeby the inventor, coatings were made with alpha-cyclodextrin which iscapable of binding relatively non-polar drugs and is well known as adrug vehicle. Further, particles were prepared which were first used toadsorb tritiated dexamtethasone and then to adsorb WGA. These particleswere finally coated with dextran or bovine serum albumin to cover unusedreactive sites on the particle surface. Preparations of this sort weresubjected to repeated washing, ultrafiltration, and N-acetylglucosamineaffinity purification. These steps demonstrated a very slow release ofthe labelled dexamethasone, but also showed that the affinity purifiedparticles carried with them a high concentration of bound dexamethasone.

Particles of this sort can be used to introduce this helpful steroiddrug into neurons for delivery to areas of spinal cord injury. As hasalso been shown by the inventor, such particles are extruded from theneuron into the endoneurial space and so are then placed in a positionto properly interact with and activate cell surface glucocorticoidreceptors at otherwise inaccessible locations inside the blood brainbarrier.

An entirely novel means of producing metal oxide particles under mildconditions suitable for delicate proteins and peptides has also beendeveloped by the inventor. This method is based on the tendency of mixediron salts to commence-precipitation when the pH is raised above 4.0.Thus, rather than raising the pH of the solution to pH 9, 10 or 11 as isdone in all previously known methods, the new method involvesprecipitating the crystals at physiological pH in biochemicallytolerable buffer solutions.

Thus viewed from a further aspect the invention provides a method ofproducing a physiologically tolerable particulate metal oxide, saidmethod comprising precipitating said oxide from a biologicallytolerable, physiological pH buffered solution, optionally containing acoating agent whereby coated particles are formed.

For instance, a strong buffer such as 1 molar HEPES pH 7.4 is preparedwith the desired coating molecules in the buffer solution. The mixedmetal sa t solution, either with or without dextran, is then added tothe buffer solution in dropwise fashion. This method has resulted in theproduction of stable coated colloids produced at physiological pH. Thisnew method of production of these crystals greatly broadens the range ofpharmaceuticals which can be included in the particle coat of these10-100 nm particles for delivery by intraneural or other routes.

Treatment of HTLV-I Associated Myelopathy: A focal infection of thespinal cord associated with HTLV-1 (Human T-Cell Leukaemia Virus,type 1) in which there is involvement of the phagocytic microglia andoligodendrocytes as well as of neurons has presented a very forbiddingpicture for any possible treatment options. What few anti-viral agentshave been available have poor penetration of the blood brain barrier andso must be administered intravenously in very high concentration toachieve potentially therapeutic dose levels in the spinal cord.Similarly, intrathecal administration into the CSF allows uncontrolledspread of the anti-viral agent throughout the CNS, affecting varioussites of reduced blood brain barrier (e.g. median eminence of thehypothalamus, pineal gland, and area prostrema) more than the infectedsite. Further, intrathecally administered drugs tend to be swept out ofthe CSF into the blood stream over a relatively rapid time coursecompared to what is required for anti-viral therapy.

However, because of the focal nature of the lesion, it is quite easy toidentify the dermatome supplied by the involved location of the spinalcord. Therefore, by undertaking intramuscular injections in neck/backmuscles as well as in muscles placed at greater distances from thespinal cord, it is possible to set up a continuous inflow of specificmedication into the CNS neurons, and by transcellular transport into thesurrounding oligodendrocytes and microglia. The active agents used caninclude small molecules conjugated to a nerve adhesion molecule, smallliposomes or other types of nanoparticles carrying antiviral drugs. Theadministration of steroids by the same route can also help to reduceinjury due to inflammatory components of the disease.

Where transport in the affected dermatome is severely impaired byextensive cell death, injections on the contralateral side of the bodyand at dermatomes below and above those affected will still bring thedrugs into a relatively high concentration at close proximity to thelesion and usefully across the blood brain barrier.

Pain: Among the most common of all tasks faced by the physician orsurgeon is the treatment of a severe localized pain whose duration willprobably be only a few days but which will cause considerable distressand discomfort to the patient. This might be from a bone fracture, anklesprain, a surgical incision, abscess, severe back muscle spasm,exacerbation of arthritis, dental extraction, burn, or tumour amongother problems. The available pharmaceutical options at present fallinto four main categories, an oral drug such as aspirin or acetaminophenwhich acts at the site of injury by altering the effects of prostacyclinrelated pain mediators, arrange of oral or locally injectedanti-inflammatory drugs of both steroidal and non-steroidal composition,locally injected anaesthetics such as marcaine or lignocaine whichreduce neural activity, and the systemic administration of opiates oropiate analogs. by oral, intramuscular or intravenous routes. There area few extraordinary treatments most commonly undertaken in cancerpatients such as the placement of continuous infusion morphine cannulasnear the spinal cord but these are little used because of difficulty inpreventing infection and the complexity of placing them. Further, suchtreatments cannot be used for pain in the arm or neck because freeopiates in spinal fluid of the upper spinal cord may reach the medullaand cause respiratory arrest.

If opiate or anti-inflammatory drugs are included in particles, bound tonerve adhesion molecules, and then injected in the dermatome/myotomewhich is involved in the pain, then an extremely efficient distributioncan be achieved. This permits the extended administration of an opiateat an extremely high concentration in the dorsal root ganglion anddorsal root entry zone of the spinal cord at the involved level, whileresulting in negligible systemic levels of the drug. For opiates, thisavoids tolerance, sedation, respiratory depression and addiction andprovides for steady long term administration over days after a singleinjection. For steroids and non-steroidal anti-inflammatory drugs, thiswill help reduce the risk of gastric irritation and internal bleeding aswell as the other side-effects of steroids.

Some types of chronic pain syndromes are remarkably resistant to allstandard pain medications including the various opiates and opiatederivatives. The prototype for this sort of pain is trigeminal neuralgiaand indeed the sine qua non of this syndrome is that the pain can berelieved only by anticonvulsant medications such as carbamazepine. Thissort of chronic pain is often viewed as a kind of focal sensoryepilepsy. The problem in treating such pain is that there is often greatdifficulty in achieving adequate doses at the dorsal root entry zone ofthe spinal cord or the trigeminal nuclei in the brainstem withoutcausing unacceptable systemic side effects. By use of pharmaceuticalagents according to the invention, agents such as carbamazepine can bedelivered via an intraneural route after intramuscular injection orintradermal injection.

Spinal Cord Injury: Although acute spinal cord injury has long provenvery difficult to treat, it has recently been appreciated that extremelyhigh doses of methylprednisolone given intravenously over the first 24to 48 hours post-injury can be significantly beneficial to eventualneurological outcome. Of course, the actual site of action is at theinjury location in the spinal cord, but the dose is limited by theextremely large amounts of steroid which must be distributed to the restof the body for no useful purpose.

These steroids can be incorporated into liposomes or other particles ofappropriate size, conjugated to a nerve adhesion molecule and injectedintramuscularly in back or neck muscles at the level of sensory or motorloss. When this is done, they are transported directly into the nervoussystem at a continuing flow and arrive precisely at the site of injury.Wherever actual nerve compression or injury has occurred, thetransported agent will accumulate and automatically achieve particularlyhigh concentrations. Drug leaking out of torn or inflamed cells, andextruded into the nervous tissue around the affected neurons will act onother injured tissues in the vicinity. This will also assure gooddelivery into the spinal cord grey matter even when the fracture hascaused venous congestion which is slowing vascular delivery to the mostaffected areas.

Certain pharmaceutical agents useful in the method according to theinvention are already known—others may be produced by methods analogousto those used for producing the known agents, e.g. for particulateagents: obtaining particles of a matrix material comprising aphysiologically active agent or diagnostic marker; optionally coatingsaid particles with a physiologically tolerable optionally biodegradablecoating material, e.g. a natural or synthetic polymer or derivativethereof such as latex, polylactic acid, proteins, albumin,polysaccharides, starches, dextrans, polymerized sugar alcohols, etc(see for example EP-A-184899 (Jacobsen)), and conjugating said particle(optionally via coupling to a said coating, optionally after appropriatederivatization thereof e.g. to provide a binding site or to block excessbinding sites) to a nerve adhesion molecule, preferably with a NAM:particle ratio of up to 10, especially up to 5 more especially up to 2and most preferably about 1; optionally separating NAM-conjugatedparticles so formed from unconjugated particles, preferably by sizeseparation, especially preferably by repeated size separation followedby at least one affinity separation; optionally sterilising theNAM-conjugated particles, if desired after formulation thereof with apharmaceutical carrier and optionally with further conventionalpharmaceutical excipients, e.g. viscosity enhancing agents, pHregulators, osmolality adjusting agents, etc.

The matrix material used may be an inorganic matrix, e.g. a metal oxide,or an organic matrix, e.g. a polymer such as a cross-linked starch ordextran, and it may serve as a carrier for the physiologically activeagent or diagnostic marker or it may itself serve as the active agent ormarker, as would for example be the case with superparamagnetic ferritecrystals.

Incorporation of the agent or marker within a carrier matrix can beachieved by conventional techniques, for example by co-precipitation, bysteeping a porous matrix material to impregnate it with the desiredagent or marker, by exposing the agent to ultrasonically suspended,uncoated metal oxide particles, or by means of the bufferedprecipitation technique described herein.

The matrix particles should desirably be relatively uniformlydimensioned, e.g. within the ranges discussed above, and this may beachieved for example by conventional screening or particle precipitationtechniques. Monodisperse particles will be preferred.

Where the agent used according to the invention is non-particulate itmay again be produced by conventional techniques, e.g. by binding adesired nuclide directly or via a chelant molecule to a NAM or bybinding a chromophore or fluorophore or a physiologically activemolecule to a NAM, optionally and indeed preferably so as to provide abiodegradable bonding which will permit liberation of thephysiologically active agent after endocytosis.

For axonal delivery of therapeutic or prophylactic substances, incertain cases it may be desirable in the method of the invention toselect physiologically active substances which occur naturally inneurons or which are analogues of such naturally occurring substances.

Viewed from a further aspect the invention provides a process for thepreparation of a particulate pharmaceutical agent according to theinvention which process comprises conjugating a NAM to an optionallycoated particulate physiologically active or diagnostically markedsubstance.

Viewed from a yet still further aspect the invention provides a processfor the preparation of the physiologically tolerable marked metaloxides, metal sulphides or alloys of the invention which comprisesprecipitating a said metal oxide or sulphide from a solution containinga positron emitter nuclide and preferably also containing an elementhaving high positron affinity, and if desired reducing said precipitate.

Viewed from a yet still further aspect the invention also provides aprocess for the preparation of the modified spinel and garnet particlesaccording to the invention which process comprises precipitating di andtrivalent metal ions of ionic radii such as to permit crystals of spinelor garnet structure to form, said precipitation being from a solutioncontaining scandium, a radioactive yttrium isotope, a sixth periodmetal, a high MR receptivity nuclide or an element having a desiredtherapeutic or prophylactic activity.

In these particle precipitation processes according to the invention thephysiologically active, marker, or high positron affinity elements to beincorporated into the particles may themselves be in solution oralternatively they may be in fine “seed” crystals which become includedin the precipitating particles.

For administration in vivo, the dosages used will clearly depend upon awide range of factors such as the patient's weight, the specificity ofthe NAM (for NAM-conjugated agents), the nature of the imaging orvisualization modality (e.g. ultrasound, MRI, CT, PET, scintigraphy,etc) where the agent is to be used to assist surgery or diagnosticinvestigations, the nature of the physiologically active or diagnosticmarker component of the pharmaceutical agent, the extent or severity ofthe injury or ailment that is being investigated or treated, thedistance over which axonal transport is required, etc. The appropriatedosage however can readily be determined taking these factors intoaccount.

However the intramuscular administration according to the invention ofNAM-targetted agents offers the possibility of very efficient and veryspecific delivery. Thus taking the example of a PET contrast agent, tofill the volume of a peripheral nerve one might require 0.1 microcurie,i.e. 0.01 μCi/ml. The injection site might commence with 50 μCi in a 10ml volume of muscle (5 μCi/ml), but within 24 hours the injectate of asmall molecule tracer with minimal affinity for muscle would distributein about 50 liters of blood and extracellular fluid space yielding aconcentration of 0.001 μCi/ml and thus even reconcentration in forexample liver or kidney would not overwhelm the signal from the nerve.

For MRI, NAM-targetted 10K dextran-coated superparamagnetic ferrousferrites, e.g. incorporating Zn(II) and Mn(II) in normal spinelinclusions to enhance magnetization and optionally Co or Mn but muchmore preferably Sc doped to permit detection/verification by MRS, mayconveniently be administered intramuscular as 10-100 μl doses containing5-20 mg Fe/ml, e.g. produced by a Centriprep-30 concentrator. 10000 MWdextran may be replaced by 1500 MW or more preferably 6000 MW dextran.

The invention is illustrated in more detail by the following Example ofdiagnostically marked ferrites.

EXAMPLE

Ferrite particle synthesis can be efficiently carried out in less than24 hours. The chloride salts of the metals with the positron nuclide (ifdesired) at specific activities of 1 μCi-1 mCi/mM Fe (370 MBq-3.7GBq/μM) of 2+ and 3+ oxidation state metal (both metal salts may bestable Fe⁵⁶) are dissolved in a saturated or supersaturated solution of1,500 to 10,000 MW dextran preferably 10,000 MW in a ratio nearMt(II)1.0:Fe(III) 2.0 at a concentration of 0.2 to 1.0 molar and at atemperature of 0-60° C. depending upon the final particle sizedistribution desired but preferably at 50° C. and where Mt is thedivalent cation of a transition metal or of a mix of transition metals.Typical starting amounts are 540 mg FeCl₃, 230 mg FeCl₂, 3 gm Dextran10K, in 4.5 ml of dH₂O. The dextran solution should be heated onlybriefly to avoid recrystalization or sludging.

Trivalent cations (such as Sc(III)) may be used in low ratios if theyare stoichiometrically balanced with monovalent metal salts, preferablyLiCl. The ferrites are precipitated by addition of 5 to 10%, preferably7.5% aqueous solution of NH₃ to reach a pH of 9 to 12 and preferably-pH11 (about 15 ml added to 7.5 ml of dextran/metal salt solution). Thissolution can be heated to 60° C. prior to adding it to the metal/dextransolution.

By omitting LiCl in the precipitation reactions with ₂₅Mn⁵², and ₂₆Fe⁵²,any chromium decay product is unlikely to remain at a II oxidation stateand so will tend to be excluded from the crystal, resulting in a sort offinal purification at time of synthesis which effectively increases thespecific activity of the nuclide and helps preserve maximal possiblemagnetic saturation of the ferrites.

A variety of sizes of dextrans can be used, for example ranging from1.5K to 40K MW although the 10K dextrans have proven most reliable inthese syntheses. Changes in outer coating also effect the tumblingbehaviour of the particles and this can have an effect on some resonantbehaviour of the particles and on their interactions with watermolecules. It is also possible to coat the particles withnon-metabolizing latex from for example cyanoacrylate monomers to altertheir rate of processing through the cells. Other biodegradable coatingssuch as polylactic acid or even protein/albumin coats can be applied. Ashift in average crystal core size towards smaller size can be producedby lowering the temperature of the synthetic reaction or elevating thepH. However, a variety of separation techniques may then be required totrim the size distribution to select the desired size range.

Additionally, the spinel crystal can be constituted of mixed metals invarious amounts in order to achieve various specific optimizations.Mixed spinels including various useful transition series metals, andeven some lanthanide metals can be made by adding the metal chloridepowders directly to the saturated dextran solution prior to alkaliprecipitation.

The product of the reaction is centrifuged 2 times at 1,000 g×10 minutesand one time at 1,500 g×10 minutes to remove particulates which arediscarded in the precipitate. The resulting suspension is passed througha 2.5 cm×40 cm column of Sephadex G-25M/150 {circle around (R)}(Pharmacia) equilibrated in 0.1M NaAcetate buffer pH6.5 in order toremove free metal ions, particulates, ferrous hydrous oxides, chlorideand ammonia.

The Sephadex eluant is then passed through successively finermicrofilters. Two passes through a 0.22 micron nylon filter are followedby two passes through a 0.1 micron nylon filter. The third filtration isslow but can be accomplished with 100 mm or 47 mm diameter filters on asuction funnel using a 50 nm filter such as Millipore {circle around(R)} VMWP-04700 Cellulose MF filters although nylon filters arepreferable. The speed and general success of this step are highlydependent on the initial precipitation conditions being most efficaciouswith smaller particle size distribution. These filtrations may also beaccomplished with centrifugal filters.

This is cleared, desalted, and size trimmed product is then concentratedwith a Centriprep-30 {circle around (R)} (Amicon) ultrafilter, at 1,500g for 45 minutes, to achieve a final volume of five to seven ml. Thesample is then applied to a 2.5 cm×25 cm column of Sephacryl-200 R(Pharmacia) equilibrated with 0.1M NaAcetate buffer pH6.5 with elutionby the same buffer. This traps dextran and small ferrous hydrous oxideswhile letting the particles pass in the excluded, unfractionated volume.The late tail of this fraction should be discarded as it contains muchof the hydrous oxide. The resulting eluant is concentrated to 4 ml witha Centriprep-30 concentrator (1,500 g for 15 minutes) for conjugation.

The particle sample in a volume of 4 ml is oxidized adding slowly 1 mlof 20 mM NaIO₄ at 23° C. This mixture is reacted while stirring(non-magnetic stirring only) for 60 minutes in the dark.

The periodation reaction is halted by passing the sample through twoPD-10 Sephadex G-25M/150 columns equilibrated with 20 mM NaBorate bufferpH8.5, concentrating with a Centriprep-30 ultrafilter to 1-2 ml thenpassing the sample through a third PD-10 column of Sephadex G-25M/150 tocompletely remove any unreacted periodate. The final volume is broughtup to 4 ml with borate buffer.

A protein solution is prepared having 2-10 mg of antibody, lectin,growth factor, or other selective adhesion molecule dissolved in 1 ml of20 mM NaBorate buffer, pH8.5. Where possible, blocking molecules toprotect the active/recognition site should be added at this point if theblocker will not be bound by the periodate activated dextran. Forexample, adding 1 mM CaCl₂/MnCl₂ helps protect the binding site on somelectins. This solution is then added to the particle solution, mixed,and allowed to incubate for 4 to 12 hours depending-upon the moleculeinvolved and the number of adhesion molecules desired per particle. Thereaction is quenched by the addition of 200 microliters of 0.5M glycinewith an additional two hours of incubation.

The covalent bonds are then reduced by the addition of 0.5 ml of 0.25MNaBH₄ with allowance for the generation of H₂ gas. After one hour ofreaction, the mixture is passed through three PD-10 columns of SephadexG-25M/150 equilibrated with 20 mM HEPES buffer at a pH of 7.4 to removeglycine, NaBH₄ and H₂, then concentrated to a 1-2 ml volume with aCentriprep-100 concentrator (500 g for 60 minutes) to clear unboundadhesion molecule and smaller, unconjugated particles. This product isthen applied to a 1.6×35 cm column of Sephacryl 200 and eluted with 20mM HEPES-buffer at pH 7.4. This column run further removes unboundtargeting molecules and traps any newly formed hydrous oxides. Theeluant is collected and concentrated with a Centriprep-100 concentratorat 500 g×30 minutes to achieve a final volume of 4 ml.

The four ml of reaction product are then applied to a 4 ml column ofaffinity ligand Sepharose 6B with divinyl sulfone links (such as SigmaA2278 for some lectins) equilibrated with 20 mM HEPES buffer pH 7.4. Itis preferable to avoid conditions normally intended to maximize bindingas this may make it impossible to elute the specific fraction. Thecolumn is then washed extensively with four to five volumes of bufferand then a 2 ml volume of 1 molar affinity eluant in the same buffer isapplied. This elutes the active fraction in a fairly sharp band.

The specific fraction is collected and passes through a PD-10 SephadexG-25M/150 column to help clear affinity eluant and then concentrated to1 mL with a small volume Centricon-30 centrifugal concentrator (1,500g×20 minutes). This product is passed through a second PD-10 column andthe final output then concentrated to a volume of 300 to 500 microliterswith a Centricon-30 concentrator (1,500 g×60 minutes). The final productis then sterilized by 0.22 or 0.1 micron filtration using a Costar 1 mlcentrifugal microfilter and stored for use.

For axon transport studies, small injections of 100-200 microliters with0.5 to 1 mg of particles (at 0.5 to 10 mCi (18.5 to 370 MBq) for PET)are made into muscle with subsequent study with positron emissiontomography, X-ray CT, magnetic resonance imaging, or magnetic resonanceheteronuclear spectroscopy one to five days after administration asindicated. For tumour evaluation, unlabelled and cold ferrite conjugatedirrelevant antibody is administered intravenously followed byintravenous administration of 0.5 to 10 mCi (18.5 to 370 MBq) ofpositron ferrite/antibody complex. Studies can then be undertaken fortumour evaluation two to five days after intravenous administration withany or all of PET, CT, MRI or MRS.

FIG. 23 shows MR images obtained with ⁵²Mn doped ferrite particlesobtained in a similar manner. A phantom was prepared using a 2 cmpolyacrylamide gel containing the particles with a 1 mm channel ofpolyacrylamide gel containing the particles at about 25 times higherconcentration. This phantom thus mimics the occurrence of a nerve insurrounding tissue (e.g. a leg). The concentrations were selected tosimulate the results of the Fe⁵⁹-WGA-ferrite study of FIG. 14.Additionally a syringe with a 3 mm diameter was taped to the outside ofthe gel-containing 2 cm diameter universal tube. As shown in FIG. 23,using a low resolution multiwire proportional position emissiontomography (MUPPET), camera it was possible to distinguish the “nerve”from both the “leg” and the syringe about 1 cm away.

The ferrite particles can be obtained by similar procedures, e.g.:

a) Following a method analogous to that of Molday (J. Immunol. Meth.52:353-367 (1982)) metal chloride powder is added directly to asupersaturated 10K dextran solution prior to precipitation with NH₄OH.Particle size separation is effected on Sephacryl 1000 with subsequentdensity gradient centrifuging.

b) The ferrite particles are synthesized by a modification of the methodof Molday (supra) which can be efficiently carried out in less than 24hours. The chloride salts of the metals with the positron nuclide atspecific activities of 10-100 mCi/μM (370 MBq-3.7 GBq/μM) of 2+oxidation state metal are dissolved in a supersaturated solution of10,000 MW dextran in a ratio near Mt(II)1.0:Fe(III) 2.0 at aconcentration of 0.5:1.0 molar and at a temperature of 20-60° C.depending upon the final particle size distribution desired and where Mtis the divalent cation of a transition metal or of a mix of transitionmetals. The ferrites are precipitated by addition of 8% aqueous solutionof NH₃ to reach a pH of 11 (about 4 ml added to 2 ml of dextran/metalsalt solution), centrifuged at 1,000 g to remove particulates, separatedand concentrated with a Centriprep-30 (Amicon) concentrator at 2,000 gfor collection of small particles in the filtrate when desired.

The products of this concentration/separation step, either filtrate(reconcentrated with Centriprep-10 concentrator) or retentate, arepassed through a preparative column of Sephadex G-25M.(150) equilibratedin 0.1M NaAcetate buffer pH6.5 at least four times the volume of theapplied sample in order to remove free metal ions, chloride and ammonia.

This desalted sample is again concentrated with aCentriprep-30-concentrator (2,500 g for one hour) to a 3-4 ml volumethen passed through a 2.5 cm×25 cm column of Sephacryl-300 (Pharmacia)equilibrated with 0.1M NaAcetate buffer pH6.5 with elution by 0.1MNaAcetate/0.15M NaCl buffer pH6.5 and 0.15M NaCl to separate unbounddextran, and the resulting fraction concentrated to 4 ml with aCentriprep-30 concentrator (2,500 g for 15 minutes) and activated byreacting with 1 ml of 20 mM NaIO₄ at 23° C. while stirring (non-magneticstirring only) for 60 minutes in the dark.

The periodation reaction is halted by passing the ferrite sample througha Sephadex G-25M (150) column equilibrated with 20 mM NaBorate bufferpH8.5, concentrating with Centriprep-30 to 1-2 ml then passing thesample through a second column of Sephadex G-25M(150) to completelyremove any unreacted periodate. The protein solution of 2-10 mg ofantibody, lectin, growth factor, or other selective adhesion moleculedissolved in 1 ml of 20 mM NaBorate buffer pH8.5 is then added to theferrite solution, mixed, and allowed to incubate for 4 to 12 hoursdepending upon the molecule involved and the number of adhesionmolecules desired per ferrite particle. The reaction is quenched by theaddition of 200 microliters of 0.5M glycine with additional two hours ofincubation.

The covalent bonds are then reduced by the addition of 0.5 ml of 0.25MNaBH₄ with allowance for the generation of H₂ gas. After one hour ofreaction, the mixture is passed through a column of Sephadex G-25M(150)equilibrated with 20 mM HEPES buffer at a pH of 7.4 to remove NaBH₄ andH₂, concentrated to a 1-2 ml volume with a Centriprep-30 concentrator(2,500 g for 30 minutes) and applied to a 1.5 cm×40 cm column ofSephacryl-300 equilibrated with 20 mM HEPES buffer pH7.4 for subsequentelution with 20 mM HEPES/0.15M NaCl buffer pH7.4 in order to removeunbound adhesion molecules and passaged into. 0.1M phosphate bufferpH7.4 via Sephadex G-25M for administration.

The resulting fraction can then be concentrated to a 1 ml volume with aCentriprep-30 concentrator for use or further purified with affinitychromatography and subsequent concentration when necessary.Reconstitution after freeze drying can also be used if desired.

c) The ferrite particles are synthesized by a modification of the methodof Molday (supra) which can be efficiently carried out in less than 24hours. The chloride salts of the metals with the positron nuclide (ifdesired) at specific activities of 10-100 mCi/μM (370 -MBq-3.7 GBq/μM)of 2+ and 3+ oxidation state metal (both metal salts may be stable Fe⁵⁶)are dissolved in a supersaturated solution of 1,500 to 10,000 MW dextranpreferably 6,000MW in a ratio near Mt(II)1.0:Fe(.III)2.0 at aconcentration of 0.2 to 1.0 molar and at a temperature of 20-60° C.depending up on the final particle size distribution desired butpreferably at 50° C. and where Mt is the divalent cation of a transitionmetal or of a mix of transition metals. Typical starting amounts are 540mg FeCl₃, 230 mg FeCl₂, 3 gm Dextran 10K, in 4.5 ml of dH₂O. The dextransolution should preferably be heated only briefly to avoidrecrystallization or sludging. Trivalent cations (such as V[III]) may beused in low ratios if they are stoichiometrically balanced withmonovalent metal salts, preferably LiCl. The ferrites are precipitatedby addition of 5 to 10%, preferably 7.5% aqueous solution of NH₃ toreach a pH of 9 to 12 and preferably pH11 (about 4 ml added to 2 ml ofdextran/metal salt solution).

A variety of sizes of dextrans can alternatively be used, ranging from1.5K to 40K MW although the 10K dextrans have proven most reliable inthese syntheses.

Additionally, the spinel crystal can be constituted of mixed metals invarious amounts in order to achieve various specific optimizations.Mixed spinels including various useful transition series metals, andeven some lanthanide metals can be made by adding the metal chloridepowders directly to the saturated dextran solution prior to alkaliprecipitation.

The product of the precipitation reaction is centrifuged 3 times at1,000 g to remove particulates which are discarded in the precipitate.The resulting suspension is passed through a preparative column ofSephadex G-25M/150 {circle around (R)} (Pharmacia) equilibrated in 0.1MNaAcetate buffer pH6.5 at least five times the volume of the appliedsample in order to remove free metal ions, chloride and ammonia.

This cleared and desalted product is then concentrated with a Centriprep100® (Amicon) ultrafilter, at 1,500 g for two hours, resuspended andagain concentrated to a 4 ml volume. This yields good clearance ofparticles below 5 nm and of unbound dextran into the filtrate fordiscard and this is the preferred method for the superparamagneticagent.

When a range of particle sizes including smaller particles are to beprocessed, this concentration step is done with a Centriprep-30concentrator. In this case, the unbound dextran will have to be removedby applying the sample as a 3-4 ml volume to a 2.5 cm×25 cm column ofSephacryl-200 {circle around (R)} (Pharmacia) equilibrated with 0.1MNaAcetate buffer pH6.5 with elution by 0.1M NaAcetate/0.15M NaCl bufferpH6.5 and 0.15M NaCl. The resulting fraction concentrated to 4 ml with aCentriprep-30 concentrator (2,500 g for 15 minutes) for conjugation.

When only very small particles are desired, the initial concentration isdone with a Centriprep-100 ultrafilter, but it is the filtrate which isthen processed further. This filtrate is reconcentrated three times witha Centriprep-30 ultrafilter to clear the dextran.

When primarily larger particles (in the-50 to 300 nm range) are desired,the desalted, ultrafiltered sample is concentrated with a Centriprep-100concentrator (2,500 g for one hour) to a 4 ml volume and then applied toa 2.5 cm×25 cm column of Sephacryl-400 R (Pharmacia) equilibrated with0.1M NaAcetate buffer pH6.5 with elution by 0.1M NaAcetate/0.15M NaClbuffer pH6.5 and 0.15M NaCl. The resulting fraction concentrated to 4 mlwith a Centriprep-30 concentrator (2,500 g for 15 minutes) forconjugation.

Particularly for the intraneural agents, it is preferable for theparticles to be less than 50 nm in diameter. Therefore, the Centriprep100 or other product from step 3 is passed through first 0.2 micron andthen 0.1 micron Nalgene {circle around (R)} nylon microfilters. Theresulting product is then concentrated to a 2 ml volume and applied to a2.5 cm×50 cm column of Sephacryl-1000 {circle around (R)} (Pharmacia)for size fractionation. Particles in the later fractions are collectedfor further processing.

The particle sample in a volume of 4 ml is oxidized adding slowly 1 mlof 20 mM NaIO₄ at 23° C. This mixture is reacted while stirring(non-magnetic stirring only) for 60 minutes in the dark.

The periodation reaction is halted by passing the sample through aSephadex G-25M (150) column equilibrated with 20 mM NaBorate bufferpH8.5, concentrating with a Centriprep-30 ultrafilter to 1-2 ml thenpassing the sample through a second column of Sephadex G-25M(150) tocompletely remove any unreacted periodate. The protein solution of 2-10mg of antibody, lectin, growth factor, or other selective adhesionmolecule dissolved in 1 ml of 20 mM NaBorate buffer pH8.5 is then addedto the particle solution, mixed, and allowed to incubate for 4 to 12hours depending upon the molecule involved and the number of adhesionmolecules desired per particle. The reaction is quenched by the additionof 200 microliters of 0.5M glycine with an additional two hours ofincubation.

The covalent bonds are then reduced by the addition of 0.5 ml of 0.25MNaBH₄ with allowance for the generation of H₂ gas. After one hour ofreaction, the mixture is passed through a column of Sephadex G-25M(150)equilibrated with 20 mM HEPES buffer at a pH of 7.4 to remove NaBH₄ andH₂, concentrated to a 1-2 ml volume with a Centriprep-100 concentrator(1,500 g for 60 minutes) to clear unbound adhesion molecule and smaller,unconjugated particles. This product can then be passaged into 0.1Mphosphate buffer pH7.4 via Sephadex G-25M for administration, or furtherpurified by affinity chromatography on non-porous beads or Nalgene{circle around (R)} affinity membranes.

The resulting fraction can then be diluted to 20 ml in sterile bufferand passed through a 0.2 micron or preferably 0.1 micron microfilter toassure sterilization. The final product is concentrated to a 1 ml volumewith a Centriprep-100 concentrator for use. Reconstitution after freezedrying can also be used to achieve desired concentrations for somepreparations.

Alternatively the product of the precipitation reaction is centrifuged 2times at 1,000 g×10 minutes and one time at 1,500 g×10 minutes to removeparticulates which are discarded in the precipitate. The resultingsuspension is passed through a 2.5 cm×40 cm of Sephadex G-25M/150{circle around (R)} (Pharmacia) equilibrated in 0.1M NaAcetate bufferpH6.5 in order to remove free metal ions, particulates, ferrous hydrousoxides, chloride and ammonia. The Sephadex eluant is then passed throughsuccessively finer microfilters. Two passes through a 0.22 micron nylonfilter are followed by two passes through a 0.2 micron nylon filter. Thethird filtration is slow but can be accomplished with 100 mm or 47 mmdiameter filters on a suction funnel using a 50 nm filter such asMillipore {circle around (R)} VMWP-04700 Cellulose MF filters.

This cleared, desalted, and size trimmed product is then concentratedwith a Centriprep-30 {circle around (R)} (Amicon) ultrafilter at 1,500 gfor 45 minutes, to achieve a final volume of five to seven ml. Thesample is then applied to a 2.5 cm×25 cm colum of Sephacryl-200 {circlearound (R)} (Pharmacia) equilibrated with 0.1M NaAcetate buffer pH6.5with elution by the same buffer. This traps dextran and small ferroushydrous oxides while letting the particles pass in the excluded,unfractionated volume. The late tail of this fraction should bediscarded as it contains much of the hydrous oxide. The resulting eluantis concentrated to 4 ml with a Centriprep-30 concentrator (1,500 g for15 minutes) for conjugation.

The particle sample in a volume of 4 ml is oxidized adding slowly 1 mlof 20 mM NaIO₄ at 23° C. This mixture is reacted while stirring(non-magnetic stirring only) for 60 minutes in the dark.

The periodation reaction is halted by passing the sample through twoPD-10 Sephadex G-25M/150 columns equilibrated with 20 mM NaBorate bufferpH8.5, concentrating with a Centriprep-30 ultrafilter to 1-2 ml thenpassing the sample through a third PD-10 column of Sephadex G-25M/150 tocompletely remove any unreacted periodate. The final volume is broughtup to 4 ml with borate buffer. The protein solution of 2-10 mg ofantibody, lectin, growth factor, or other selective adhesion moleculedissolved in 1 ml of 20 mM NaBorate buffer pH8.5. Where possible,blocking molecules to protect the active/recognition site should beadded at this point if the blocker will not be bound by the periodateactivated dextran. For example, adding 1 mM CaCl₂/MnCl₂ helps protectthe binding site on some lectins. This solution is then added to theparticle solution, mixed, and allowed to incubate for 4 to 12 hoursdepending upon the molecule involved and the number of adhesionmolecules desired per particle. The reaction is quenched by the additionof 200 microliters of 0.5M glycine with an additional two hours ofincubation.

The covalent bonds are then reduced by the addition of 0.5 ml of 0.25MNaBH₄ with allowance for the generation of H₂ gas. After one hour ofreaction, the mixture is passed through three PD-10 columns of SephadexG-125M/150 equilibrated with 20 mM HEPES buffer at a pH of 7.4 to removeglycine, NaBH₄ and H₂, then concentrated to a 1-2 ml volume with aCentriprep-100 concentrator (500 g for 60 minutes) to clear unboundadhesion molecule and smaller, unconjugated particles. This product isthen applied to a 1.6×35 cm column of Sephacryl 200 and eluted with 20mM HEPES buffer at pH 7.4. This column run further removes unboundtargeting molecules and traps any newly formed hydrous oxides. Theeluant is collected and concentrated with a Centriprep-100 concentratorat 500 g×30 minutes to achieve a final volume of 4 ml.

The four ml of reaction product are then applied to a 4 ml column ofaffinity ligand Sepharose 6B with divinyl sulfone links (such as SigmaA2278 for some lectins) equilibrated with 20 mM HEPES buffer pH7.4. Itis preferable to avoid conditions normally intended to maximize bindingas this may make it impossible to elute the specific fraction. Thecolumn is then washed extensively with four to five volumes of bufferand then a 2 ml volume of 1 molar affinity eluant in the same buffer isapplied. This elutes the active fraction in a fairly sharp band.

The specific fraction is collected and passes through a PD-10 SephadexG-25M/150 column to help clear affinity eluant and then concentrated to1 ml with a small volume Centricon-30 centrifugal concentrator (1,500g×20 minutes). This product is passed through a second PD-10 column andthe final output then concentrated to a volume of 300 to 500 microliterswith a Centricon-30 concentrator (1,500 g×60 minutes). The final productis then sterilized by 0.22 or 0.1 micron filtration using a Costar 1 mlcentrifugal microfilter and stored for use.

The invention is also further illustrated by the accompanying g drawingsalready mentioned above in which:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of one face of a spinel crystal demonstrating theposition of 1) oxygen atoms, 2) “A” sites for metal ions and 3) “B”sites for metal ions.

FIG. 2 is a table of data on various elements discussed herein arrangedin the form of the periodic table. Data shown for various elementsinclude radioisotope disintegration pattern, half life and energyprofile; magnetic resonance receptivity relative to hydrogen, Larmorfrequency at 4.7 Tesla, typical oxidation state and ionic radios, andcharacteristic positron affinity.

FIG. 3 is a schematic demonstration of the benefits of spinel moderatedemitters (SMPE) showing 4) a targeting protein in free solution with achelated positron emitting atom undergoing radioactive disintegration,5) a targeting protein bound to the surface of an SMPE particle, 6) acoated SMPE particle with positron ionization tracks marked includingone positron proceeding to annihilation before exiting the particle, 7)the paths followed by the two annihilation photons, 8) the correlationangle (nearly 180°) between the two photon paths, 9) the ionizationtrack of a positron travelling in water, and 10) the site of amatter/anti-matter annihilation reaction between an electron and theexhausted positron occurring at some distance from where the initialdisintegration took place.

FIG. 4 is a CT X-ray showing a polyacrylamide gel phantom within whichseveral gel channels are doped with different contrast agents.

FIG. 5 is a schematic diagram of a portion of a human torso depicting18) the conus medullaris at the lower termination of the spinal cord and19) a motor axon which is part of a single cell nearly three feet inlength.

FIG. 6 shows the anatomy of the spinal roots with 20) a dorsal rootganglion containing sensory neurons, 21) the dorsal containing sensoryaxons, 22) the dorsal root entry zone, 23) the dorsal ramus carryingmotor and sensory fibers to the back muscles, 24) the ventral ramuscarrying motor and sensory fibers to the limbs and anterior portion ofthe body, 25) the ventral root carrying motor axons, and 26) the ventralgrey matter of the spinal cord containing motor neuron cell bodies.

FIG. 7 depicts a motor unit omprising 27) one of several muscle cellswhich fire in unison, 28) a muscle made up of many motor units such asthe one shown, and 19) a motor axon supplying the motor unit.

FIG. 8 demonstrates 27) a myocyte or muscle cell upon which 19) a motoraxon terminates and 29) a muscle spindle with 30) sensory endings aswell as intrafusal motor innervation not shown.

FIG. 9 is a two part schematic of a peripheral nerve including 31) theepineurium sheath, 32) a fascicle, 33) the endoneurial space of afascicle, 34) the perineurium surrounding the endoneurial space andwhich is the site of the blood/nerve barrier to small moleculediffusion, 19) a motor axon seen in cross section, 35) a schwann cellsurrounding an enlarged single axon, 36) a mitochondrion within the axonseen in cross section, 37) the axolemma or membrane of the axon, 38) amicrotubule within the axon and dimension marks indication the 20 microndiameter of the motor axon.

FIG. 10 shows an axon in longitudinal section with 35) a schwann cellsheath, 39) a Node of Ranvier between two schwann cells, and 38)microtubules within the axon (19).

FIG. 11 depicts the mechanics underlying axonal transport based on arelatively stationary microtubule (38) with 40) one of a series ofmolecules of dynein and 41) one of a series of molecules of kinesin, inwhich 42) a lipid vesicle is being transported in 43) a retrogradedirection toward the cell body and 44) the anterograde direction.

FIG. 12 demonstrates some of the parts of an axon terminus with 38) amicrotubule, 42) a vesicle, 36) a mitochondrion, 46) the muscle cellmembrane, 47) a 20 to 50 nm dextran coated ferrite particle, 48) thesynaptic cleft, 49) a cell surface receptor, 50) a cell surface markeror antigen, 51) a vesicle containing an internalized group of receptorligand complexes, and 45) the diameter of the axon which is 2 to 10microns.

FIG. 13 is a graph depicting the results of the ¹²⁵I-WGA distributionstudy in which the vertical axis gives counts per minute per gm oftissue normalized by dividing by the cpm/gm of blood for the individualanimal in the series, various tissues are displayed along the long Yaxis and lines 1 through 8 reflect the results from the differentanimals with varying treatment/survival times and doses. DRG signifiesSpinal roots and dorsal root ganglia and demonstrates concentrations5-10 times higher than any other tissue except the muscle and lymphnodes near the injection site which are not shown.

FIG. 14 is a graph depicting the results of a ⁵⁹Fe WGA-dextran magnetitedistribution study after intramuscular injection. Counts per minute/gmtissue show concentration in distal and proximal ipsilateral peripheralnerve which are 50 to 100 times higher than in any other tissueexcluding the muscle and lymph nodes at the injection site which are notshown.

FIG. 15 is a graph showing results of T₂ measurements uponpolyacrylamide gels doped with varying concentration of variouspreparations of dextran coated magnetite. The white arrow indicates a T₂of 30 msec which would be a 40% reduction from normal T₂ of nerve andthe black arrow indicates concentrations of ferrite particles achievedin nerve equivalent and greater than 40 micrograms/ml. Theconcentrations in nerve are up to ten times higher than the amountsrequired to reduce T₂ in gel below 30 msec.

FIG. 16 demonstrates the general arrangements for in vivo MR microscopyof an intact nerve in the leg of experimental animal showing theposition before being moved into the MR magnet. Note that the leg isperpendicular to the long axis of the magnet.

FIG. 17 enlarges the view of FIG. 16 to show a surface coil (52) placedaround an incision line on the skin of the thigh.

FIG. 18 shows a cross section through the thigh with 53) the sciaticnerve and 54) the tibial nerve approaching an implanted cuff.

FIG. 19 demonstrates the silastic cuff (55) with a central channel (56)for the tibial nerve and three surrounding channels for various dopedpolyacrylamide gels used to standardize image contrast. The centralchannel is about 1 mm in diameter.

FIG. 20 includes photographs of the tibial nerve in the silastic cufffrom 57) the uninjected leg and 58) the injected side. The nerve is inthe central channel and is darker than the lower gel channels in theinjected leg but brighter in the uninjected side.

FIG. 21 is an electron micrograph of the tibial nerve of a rabbitcollected three days after intramuscular injection with WGA-dextranmagnetite. The photograph shows the thick myelin sheath and, within theaxon, small particles and large vesicles associated with the particles.

FIG. 22 is a blow up of a portion of FIG. 21 to 195,000×. Thisdemonstrates small ferrite particles along the microtubules as well assomewhat larger particles within two vesicles being transported.

FIG. 23 demonstrates the results of a positron emission tomography trialwith ⁵²Mn dextran coated ferrite particles. There is a “nerve gel” onemillimeter in diameter (59) cast within a larger “leg gel” where theratio of concentrations of the positron emitting ferrites is 25:1(nerve:leg), a relation reflecting the results of earlier distributiontrials.

The diameter of the test tube is about 2.2 cm (60) and there is a 1milliliter syringe taped to the outside which also contains concentratedS²Mn Ferrite. Cross sectional images (61) show ready distinction betweenthe two high concentration sources and this is demonstrated in twodimensional format in 62. Seen from anteriorly, the two sources canstill be distinguished.

What is claimed is:
 1. A pharmaceutical agent comprising a nerveadhesion molecule coupled to a particulate, physiologically activesubstance, wherein said agent, when contacted with a nerve terminus in avascularized, peripherally innervated tissue site or in a tissue siteinnervated by a spinal root, undergoes axonal transport followingneuronal endocytosis.
 2. The agent according to claim 1, wherein saidparticulate substance has a mean particle size of 10-50 nm.
 3. The agentaccording to claim 1, wherein said particulate substance has a spinelstructure.
 4. The agent according to claim 1, wherein said particulatesubstance is superparamagnetic.
 5. The agent according to claim 1,wherein said particulate substance incorporates radionuclides.
 6. Theagent according to claim 1, wherein said particulate substance remainsin aqueous solution when centrifuged at 2,500 g for 60 minutes.
 7. Theagent according to claim 1, wherein said particulate substance remainsin aqueous solution when centrifuged at 2,500 g for 30 minutes.
 8. Theagent according to claim 1, wherein said particulate substance remainsin aqueous solution when centrifuged at 2,500 g for 15 minutes.
 9. Theagent according to claim 1, wherein said particulate substance remainsin aqueous solution when centrifuged at 1,500 g for 2 hours.
 10. Theagent according to claim 1, wherein said particulate substance remainsin aqueous solution when centrifuged at 1,500 g for 1 hour.
 11. Theagent according to claim 1, wherein said particulate substance remainsin aqueous solution when centrifuged at 1,500 g for 45 minutes.
 12. Theagent according to claim 1, wherein said particulate substance remainsin aqueous solution when centrifuged at 1,500 g for 15 minutes.
 13. Theagent according to claim 1, wherein said particulate substance has amean particle size of 5-100 nm.
 14. The agent according to claim 1,wherein said particulate substance has a mean particle size of 8-70 nm.15. The agent according to claim 1, wherein said particulate substancehas a mean particle size of 20-30 nm.
 16. The agent according to claim1, wherein said axonal transport is anterograde transport.
 17. The agentaccording to claim 1, wherein said axonal transport is retrogradetransport.
 18. The agent according to claim 1, wherein said axonaltransport is fast transport.
 19. The agent according to claim 1, whereinsaid axonal transport is slow transport.
 20. The agent according toclaim 1, wherein said axonal transport is transneuronal transport. 21.The agent according to claim 1, wherein said axonal transport istranssynaptic transport.
 22. The agent according to claim 1, whereinsaid particulate substance undergoes axonal transport within a cranial,peripheral or autonomic nerve, a sensory ganglion, or an autonomicganglion.
 23. The agent according to claim 1, wherein said particulatesubstance undergoes axonal transport within the central nervous system.24. The agent according to claim 23, wherein said particulate substanceundergoes axonal transport within the spinal cord.
 25. The agentaccording to claim 23, wherein said particulate substance undergoesaxonal transport within the brain.
 26. The agent according to claim 25,wherein said particulate substance undergoes axonal transport within thebrain stem or olfactory tract.
 27. The agent according to claim 1,wherein said particulate substance undergoes axonal transport within theolfactory tract following intranasal administration.
 28. The agentaccording to claim 1, said agent comprising one hundred to one millionpharmaceutically active molecules per nerve adhesion molecule.
 29. Theagent according to claim 1, wherein said nerve adhesion molecule isselected from the group consisting of an antibody or fragment thereof, areceptor, an endorphin, vasoactive intestinal polypeptide, acalcitonin-related peptide, cholescystokinin, substance P, somatostatin,neuropeptide Y, a steroid, a viral fragment, a viral coat protein, abacterial toxin, a cell surface antigen, a lectin, wheat germagglutinin, an immunoadhesin, a neurotransmitter, a growth factor, anenzyme or fragment thereof that is selectively endocytosed for synapticrecycling purposes, and a cell adhesion molecule.
 30. The agentaccording to claim 1, wherein said nerve adhesion molecule istransferrin or beta-nerve growth factor.
 31. The agent according toclaim 1, wherein said nerve adhesion molecule has an affinity for amarker on a muscle cell surface.
 32. The agent according to claim 1,comprising a nerve adhesion molecule attached to a small liposome ornanoparticle which packages a hydrophilic or hydrophobic drug fordelivery to a selected neural site.
 33. The agent according to claim 1,wherein said physiologically active substance is selected from the groupconsisting of an anti-viral agent, a steroid, an opiate, opiatederivative, steroid or non-steroidal anti-inflammatory drug, and ananti-convulsant.
 34. The agent according to claim 1, wherein said nerveadhesion molecule is coupled directly to said particulate substance. 35.The agent according to claim 34, wherein said nerve adhesion molecule iscoupled to said particulate substance by carbodiimide binding.
 36. Theagent according to claim 34, wherein said nerve adhesion molecule iscoupled to said particulate substance by glutaraldehyde binding.
 37. Theagent according to claim 34, wherein said nerve adhesion molecule iscoupled to said particulate substance by biotin/avidin linkage.
 38. Theagent according to claim 1, wherein said nerve adhesion molecule iscoupled to said particulate substance by noncovalent interaction.
 39. Amethod of treating a living non-human or human body comprisingadministering an agent according to claim 1, wherein said agent isadministered into a vascularized, peripherally innervated tissue site orinto a tissue site innervated by a spinal root.
 40. The method accordingto claim 39, wherein said agent is administered into a tissue siteinnervated by cranial, peripheral or autonomic nerves.
 41. The methodaccording to claim 39, wherein said agent is administeredintramuscularly.
 42. The method according to claim 41, wherein saidnerve adhesion molecule has an affinity for a marker on a muscle cellsurface.
 43. The method according to claim 39, wherein said agent isadministered topically.
 44. The method according to claim 39, whereinsaid agent is administered topically upon or in the brain.
 45. Themethod according to claim 39, wherein said agent is administeredintravenously, intrathecally, intracisternally, sub-cutaneously,intradermally, by eyedrops or by bladder irrigation.
 46. The methodaccording to claim 39, wherein said agent is administered intranasally.47. The method according to claim 39, wherein said agent is administeredinto a tissue site having a volume of at least about ten times that of agroup of nerve cells which are to transport the agent.
 48. The methodaccording to claim 47, wherein said tissue site is muscle.